Squalene-hopene cyclase: Insight into the role of the methyl group on the squalene backbone upon the polycyclization cascade. Enzymatic cyclization products of squalene analogs lacking a 26-methyl group and possessing a methyl group at C(7) or C(11)

DOI: 10.1039/b404287e

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Organic and Biomolecular Chemistry p. 2012 - 2022 (2004)

Update date:2022-08-02

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Authors:

Nakano, Shin-Ichi

Ohashi, Shumi

Hoshino, Tsutomu

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Article abstract of DOI:10.1039/b404287e

To provide deep insight into the polycyclization reaction of squalene, some analogs were synthesized and incubated with the cell-free homogenates of the recombinant Escherichia coli encoding the wild-type squalene cyclase. The presence of C(6)-Me leads to an efficient polycyclization cascade. Substitution of the C(14)-H and the C(18)-H with a methyl group halted the polycylization reaction at the tricyclic ring stage having a 6/6/6-fused ring system and the tetracycle with a 6/6/6/6-fused ring, respectively, both of which were produced according to a Markovnikov closure. Replacement of the C(7)-H and the C(11)-H with a methyl group led to no cyclization. These results, in conjunction with our previous reports, indicated that the methyl positions are important for bringing to completion of the normal polycylization reaction and further demonstrated that the precise steric bulk size at the methyl positions of squalene is critical to the correct folding and the strong binding of the substrate to the squalene cyclase.

Full text of DOI:10.1039/b404287e

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Squalene–hopene cyclase: insight into the role of the methyl group

on the squalene backbone upon the polycyclization cascade.

Enzymatic cyclization products of squalene analogs lacking a

26-methyl group and possessing a methyl group at C(7) or C(11)†

Shin-ichi Nakano, Shumi Ohashi and Tsutomu Hoshino*

Department of Applied Biological Chemistry, Faculty of Agriculture,

and Graduate School of Science and Technology, Niigata University, Ikarashi,

Niigata 950-2181, Japan. E-mail: hoshitsu@agr.niigata-u.ac.jp;

Fax: ϩ81-25-262-6854; Tel: ϩ81-25-262-6637

Received 22nd March 2004, Accepted 28th May 2004

First published as an Advance Article on the web 29th June 2004

To provide deep insight into the polycyclization reaction of squalene, some analogs were synthesized and incubated

with the cell-free homogenates of the recombinant Escherichia coli encoding the wild-type squalene cyclase. The

presence of C(6)–Me leads to an eﬃcient polycyclization cascade. Substitution of the C(14)–H and the C(18)–H with

a methyl group halted the polycylization reaction at the tricyclic ring stage having a 6/6/6-fused ring system and the

tetracycle with a 6/6/6/6-fused ring, respectively, both of which were produced according to a Markovnikov closure.

Replacement of the C(7)–H and the C(11)–H with a methyl group led to no cyclization. These results, in conjunction

with our previous reports, indicated that the methyl positions are important for bringing to completion of the normal

polycylization reaction and further demonstrated that the precise steric bulk size at the methyl positions of squalene

is critical to the correct folding and the strong binding of the substrate to the squalene cyclase.

Introduction

Linear squalene 1 is cyclized into the pentacyclic triterpene,

hop-22(29)-ene 2 and hopan-22-ol 3 (ca. 5 : 1), which is

mediated by squalene–hopene cyclase (SHC) from prokaryotic

species (Scheme 1).^1–3This polycyclization reaction proceeds

with regio- and stereochemical speciﬁcity under precise enzyme

control to form the 6/6/6/6/5-fused ring system and nine new

chiral centers. SHC folds 1 into the all pre-chair conformation

(a product-like conformation) inside the enzyme cavity, leading

to the ﬁnal hopanyl cation through sequential ring-forming

reactions. The proton elimination occurs exclusively from the

(23Z)-methyl group, but not from the E-methyl group, to form

2.^1,4A nucleophilic attack of water molecule on the cation

aﬀords 3. Recent studies by three-dimensional X-ray crystallo-

graphic analyses⁵and by site directed mutagenesis experi-

ments^1,2have provided deeper insights into the polycyclization

mechanism. The investigations by using modiﬁed substrates

also have given important information. In previous papers,^4,6,7

we have shown that the methyl groups at the both terminal sides

play crucial roles both in initiating the polycyclization and in

constructing the ﬁve-membered E-ring,^4,6and also established

that the methyl group at C(10) of the central part has a critical

role in forming the all pre-chair structure⁷(Scheme 2). In the

case of the substrate analogs 4 and 5 lacking 25-Me and 1-Me

of 1, respectively, the polycyclization reaction started from the

isopropylidene moiety, and not from the methyl-deﬁcient side.

This ﬁnding was further conﬁrmed by bisnorsqualene lacking

Scheme 1 Polycyclization reaction of squalene 1 by hopene synthase:

1 is folded in all pre-chair conformation to give the hopanyl cation,

both 1-Me and 25-Me.^4aAs major product(s), tetrahymanol

skeletons 6 and 7 having a six-membered E-ring were con-

structed instead of the ﬁve-membered E-ring of 2.⁶When the

substrate analog 9 lacking the 27-Me of 1 was incubated, two

cyclization pathways a and b occurred (Scheme 2). Path a shows

that 9 was recognized as C(10)-norsqualene, while path b as

C(15)-norsqualene. C(10)-Norsqualene aﬀorded novel carbo-

leading to hopene 2 and hopanol 3 in a ratio of 5 : 1. The deprotonation

from the hopanyl cation gives 2, which occurs exclusively from the 23Z-

Me of 1 to give 2, while 3 is produced by a nucleophilic attack of

a water molecule to the hopanyl cation.

cyclic skeletons 10 and 11 having the 6/5ϩ5/5ϩ(6) ring system.⁷

To aﬀord the novel skeleton, 9 must be folded in an unusual

conformation,⁷completely diﬀerent from the all pre-chair con-

formation. On the other hand, C(15)-norsqualene was folded in

an all pre-chair conformation to give 12 and 13 (10.5 : 7.3) in a

† Electronic supplementary information (ESI) available: Additional

characterizatrion data for products 34–45. See http://ww w .rsc.org/

suppdata/ob/b4/b404287e/

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C-14, instead of C-10 and C-15. We describe here the enzymatic

products obtained from these analogs and discuss the role

of the branching methyl group(s) upon the polycyclization

cascade.

Results

Syntheses of analogs 14–17

Analogs 14–17 were synthesized according to Scheme 3 and

described in detail in the Experimental section. Compound 16

was synthesized by the allyl coupling of a geranylgeranyl

bromide 20 with geranyl phenylsulfone 21 as the key step. The

cleavage of the carbon–sulfur bond was done with LiBEt₃and

a catalytic amount of PdCl₂(dppp) [dppp = 1,3-bis(diphenyl-

phosphino)propane] in Et₂O.⁹Aldehyde 23, obtained from 1,

was subjected to a Grignard reaction with CH₃I to give alcohol

Scheme 2 The cyclization pathways of norsqualenes 4, 5 and 9, and

the substrate analogs 14–17 employed in this investigation. Analogs 4

and 5 lack the methyl group at the terminal double bond of 1. The

(23Z)-Me of 1 is missing for 4, while (23E)-Me is missing for 5. The

methyl group at the central part is absent in 9. The both left and right

sides have an isopropylidene moiety, thus

9 can undergo the

polycyclization reaction from the left (path a, recognized as C(10)-

norsqualene) and right sides (path b as C(15)-norsqualene). Unusual

folding conformation, completely diﬀerent from the all pre-chair

conformation shown in Scheme 1, was adopted via path a to aﬀord

10 and 11 having a novel carbocyclic skeleton of a 6/5ϩ5/5ϩ(6) ring

system.

similar manner as the formation of 2, but the stereochemistry at

C(21) of 13 was diﬀerent from those of natural type 12 and 2.⁷

Based on these experimental results, we have proposed that the

binding or accepting sites of the methyl groups at the terminal

and central sides are involved in the cyclase enzyme.

Squalene 1 is a symmetrical molecule and the methyl groups

are arranged at regular intervals on the backbone. As described

above, SHC has binding sites for the branching methyl group of

1. To provide further insight into the binding sites of the methyl

groups, analogs 14–17 were synthesized and incubated with

the wild-type SHC. The 26-Me at C-6 of 1 is absent in 14.⁸

Compounds 15–17 are squalene analogs in which methyl

groups are arranged at position(s) diﬀerent from 1 (regio-

isomers of methyl groups). Analog 15 has a methyl group at C-7

instead of C-6 of 1. Analog 16 has a methyl group at C-11

instead of C-10. Analog 17 has methyl groups at both C-11 and

Scheme 3 Synthetic scheme of squalene analogs 14–17. Reagents and

conditions: (i) PheSO₂Na/DMF; (ii) n-BuLi/THF–[(CH₃)₂N]₃PO (4 : 1);

(iii) LiBEt₃H, PdCl₂(dppp)/Et₂O at 0 ЊC; (iv) m-CPBA/CH₂Cl₂at 0 ЊC;

(v) H₅IO₆/Et₂O; (vi) CH₃I/Mg in Et₂O; (vii) CrO₃(py)/CH₂Cl₂; (viii)

(EtO)₂POCH₂CO₂Et/NaH in THF; (ix) (iso-C₄H₉)₂AlH/Et₂O; (x) PBr₃/

THF; (xi) CH₂(CO₂Me)₂/NaOEt in EtOH; (xii) 1 M KOH/MeOH, then

HCl, heat; (xiii) 5% dry HCl/MeOH; (xiv) LAH/THF; (xv) PCC/

CH₂Cl₂; (xvi) Ph₃P^ϩCH₃CHBr^ϪCO₂Et/NaOH; (xvii) pyrrolidine/n-

BuLi/CuI/Et₂O.

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24, which was then oxidized into 25 with Collin’s reagent.

Wadsworth–Emmons reaction of 25 with ethyl diethylphos-

phonoacetate gave the ester 26a, which was reduced with

DIBAL-H to aﬀord alcohol 27a and the allyl coupling of the

bromide 28a, prepared by treatment of 27a with PBr₃, with

phenylsulfone 29 gave 30a. The deprotection of the phenyl-

sulfone group from 30a was carried out with LiBEt₃H to aﬀord

the desired 15 in a similar manner as described above. Analog

14 was prepared with essentially the same method as in the

synthesis of 15, but 23 was directly subjected to Wadsworth–

Emmons reaction to give 26b. Analog 17 was synthesized

as follows. Geranyl bromide 19 was condensed with methyl

malonate to elongate by a C₂unit, followed by decarboxylation

and methylation to yield methyl ester 31. Then the following

ﬁve steps were carried out: the reduction of 31 with LAH,

hexane extracts was removed with a short SiO₂chromatography

column eluting with a mixture of n-hexane–EtOAc (100 : 20).

Fig. 1(A) shows the product distribution pattern obtained by

incubating 1. No substrate remained in the incubation mixture.

Three major products (34, 35 and 36) from 14 were found in a

good conversion ratio, as shown in Fig. 1(B). The yield of each

product was estimated by the GC (Fig. 1(B)) to be as follows:

29.4, 19.8, 34.8 and 1.4% for 34, 35, 36 and unreacted 14,

respectively. The remaining products (total yield, 14.6%)

comprised of some minor products, but the yield of each

minor product was less than 2–4%. As shown in Fig. 1(C),

many products were found from the incubation mixture of

analog 15 in a good conversion ratio (ca. 98%) and only a small

amount of 15 was recovered (1.4%). Analog 16 was converted

into 45, but in a small yield (7%, Fig. 1(D)). The conversion of

17 was low as shown in Fig. 1(E).

oxidation with PCC, the Wittig reaction with the ylide Ph P᎐

᎐

C(CH₃)CO₂Et, reduction with LAH, and bromination with

PBr₃. The C₁₅-bromide, thus prepared, was subjected to an allyl

coupling reaction by using pyrrolidine/n-BuLi/CuI,¹⁰leading to

the desired 17 after puriﬁcation by AgNO₃–SiO₂column

chromatography.

Enzymatic products 34, 35 and 36 from 14

With the cell-free homogenates (150 cm³) prepared from a 3 L

culture of E. coli encoding the native SHC, 40 mg of 14 was

incubated at optimum catalytic conditions (pH 6 and 60 ЊC).

After lyophilizing the incubation mixture, the residues were

extracted with hexane and passed through a short SiO₂column

to remove Triton X-100 by eluting with a mixed solvent of

hexane–EtOAc (100 : 20). Three major peaks 34–36 were found

in addition to some minor peaks and a small amount of

recovered 14, as shown in Fig. 1(B), but the analysis of SiO₂

TLC showed only two major spots, their R_fvalues being close

to those of 2 and 3; 0.81 with hexane for 2, 0.57 with EtOAc–

hexane = 100 : 20 for 3. Column chromatography over SiO₂

eluting with hexane gave the low polar fraction, then elution

with a mixed solvent of hexane–EtOAc (100 : 5) gave the more

highly polar product 35, which was further puriﬁed by normal

phase HPLC (hexane–iso-PrOH = 100 : 0.05). The GC analysis

showed that the low polar fraction was comprised of two

products, 34 and 36. Complete separation of 34 and 36 was

successfully done by HPLC (reverse-phased C₁₈column) with

THF–H₂O (55 : 45). The structures of 34–36 (Fig. 2) were

determined by detailed analyses of the NMR spectra including

DEPT, COSY-45, HOHAHA, NOESY, HMQC and HMBC.

Products 34 and 35 had a pentacyclic hopane skeleton lacking a

methyl group at C-18. In the HMBC spectra of 34 and 35, the

methyl signal (δ_H1.01, s, Me-25) had strong correlations for

both C-5 (δ_C56.8) and C-9 (δ_C51.3), indicating that this methyl

group is positioned at C-10. On the other hand, the proton

signal (δ_H1.26, m, H-10) of 36 had clear HMBC cross peaks for

both C-4 (δ_C33.2) and C-8 (δ_C41.0), indicating that a methyl

group at C-10 was absent in 36. The polar compound 35 had a

tertiary hydroxy group (δ_C73.9), the position of which was

determined to be at C-22 from the HMBC cross peaks of

Me-28/C-22 and Me-29/C-22. Compounds 34 and 35 were the

products when the substrate 14 was recognized as C(19)-

norsqualene (14b of Scheme 4), while 36 was produced from

C(6)-norsqualene (14a). The produced amounts of 34 and 35

were ca. three times higher than that of 36, indicating that

the SHC recognized the C(19)-norsqualene 14b over the C(6)-

norsqualene 14a as the substrate (Scheme 4).

GC analyses of the enzymatic products

Fig. 1 shows the gas chromatograms of the incubation mixtures

prepared by incubating 1 and 14–17 with the cell-free homo-

genates of E. coli clone encoding the native SHC from

Alicyclobacillus acidocaldarius. Identical incubation conditions

were employed to compare the quantities and distribution

pattern of enzymic products obtained from each substrate

analog. Incubation conditions were as follows: substrate

analog, 1.0 mg; the cell-free extract as the enzyme source,

1.5 cm³, Triton X-100, 20 mg; optimal pH, 6.0; optimal temper-

ature, 60 ЊC; incubation time, 16 h; total volume, 5 cm³. To the

reaction mixture, was added 5% KOH/MeOH and the products

were extracted with hexane. Triton X-100 included in the

Enzymatic products from substrate 15

Fig. 1(C) depicts the product distribution pattern of the reac-

tion mixture obtained by incubating 15 at optimum catalytic

conditions. Many products were detected. To isolate these

products, 100 mg of 15 was incubated for 20 h with the cell-free

homogenates (200 cm³) from a 2 L culture of the cloned E. coli.

The enzymic products consisted of a mixture of relatively high

and low polar materials. SiO₂column chromatography eluting

with a step-wise elution (100% hexane to 10% EtOAc–hexane)

aﬀorded pure 40, 43 and 44 (high polar compounds) in this

elution order, but the separation of low polar materials was

Fig. 1 Gas chromatograms of the reaction mixtures obtained by

incubating 1 (A), 14 (B), 15 (C), 16 (D) and 17 (E) with the wild-

type SHC. Triton X-100 was removed by a short SiO₂column

chromatogram.

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Fig. 2 Structures of products 34–45 obtained from analogs 14–17.

unsuccessful. Repeated washing with cold MeOH succeeded in

shown in structure 37 of Fig. 2. Product 38 also showed two

the separation of solid and oily materials. The solid fraction

consisted of two enzymic products, each of which was puriﬁed

by 10% AgNO₃–SiO₂column chromatography eluting with

hexane–EtOAc (100 : 0.02), giving pure products 41 and 42.

The mixture of oily materials contained three major products

37–39, which were successfully separated by 10% AgNO₃–SiO₂

column chromatography (EtOAc–hexane = 0.05 : 100), and

further puriﬁed by HPLC (reverse-phased C₁₈column) with

THF–H₂O (70 : 30), aﬀording pure products 37–39. The struc-

tures of these products were unequivocally determined by

detailed 2D NMR analyses. Product 37 had one oleﬁnic proton

(δ_H5.38, br t, J 6.7), correlated with δ_C125.3 in the HMQC,

and had three quaternary sp²carbons (δ_C130.9 s, C-22; 134.1 s,

C-18; 135.0 s, C-13), indicating the presence of two double

bonds. Two allyl methyl protons (δ_H1.83 and 1.74, Me-30 and

Me-29, respectively) that had the correlations with the two sp²

carbons at C-21 and 130.9 were found, suggesting that one

terminal isopropylidene moiety remained without undergoing

cyclization, and thus 37 was a tetracyclic product. One doublet

methyl signal was found (δ_H1.24, d, J 7.0, Me-28). The remain-

ing ﬁve methyl groups that appeared as singlets in the ¹H NMR

spectrum was determined to be Me-23, -24, -25, -26 and -27 by

HMBC and NOESY spectra. Strong HMBC cross peaks of

Me-27 (δ_H1.31)/C-13 and Me-28 (δ_H1.24, d, J 7.0))/C-18 have

established the position of another double bond. The clear

NOE between Me-28 and Me-26 (δ_H1.10, s) indicated that Me-

28 was arranged in β-orientation. Thus, the structure of 37 was

determined to have a 6/6/6/6-fused tetracyclic ring system, as

double bonds (δ_C117.5 d, C-7; 125.9 d, C-21; 131.0 s, C-22;

145.9 s, C-8) and two oleﬁnic protons (δ_H5.61, br s, H-7 and

5.42, br t, J 7.0, H-21) according to the NMR spectra. The two

allyl methyl groups (δ_H1.85, s, Me-30 and 1.77, s, Me-29) were

correlated with C-21 and C-22 in the HMBC spectrum. Thus,

38 also had a 6/6/6/6-fused tetracyclic ring system, but the

position of the other double bond was diﬀerent from that of 37.

Me-26 (δ_H1.22, s) had a HMBC cross peak for C-8, indicating

that the double bond position was at C7–C8. A strong NOE

between Me-26 and H-18 (δ_H1.12, m) was suggestive of

β-orientation of H-18. Me-27 (δ_H1.11, s) was oriented in

α-disposition, since a clear NOE between Me-27 and H-9

(δ_H2.38, m) was observed. A clear NOE between Me-28

(δ_H1.10, d, J 5.8) and Me-26 deﬁnitively demonstrated the

β-orientation for Me-28. Product 39 had an oleﬁnic proton

(δ_H5.43, br t, J 6.7, H-21), which was correlated with both of

two allyl methyl signals (δ_H1.84, s, Me-30 and 1.75, s, Me-29)

in the COSY-45 spectrum, suggesting that 39 also had a tetra-

cyclic structure. The ﬁndings of seven methyl signals and

the vinyl protons (δ_H4.92, s and δ_H5.05, s) in the H NMR

spectrum indicated that one of eight methyl groups involved in

15 underwent a deprotonation reaction from one methyl group.

The vinyl protons had a strong cross peak for C-16 (δ_C33.3) in

the HMBC spectrum and showed clear correlations for H-16

(δ_H2.32, 2H, dd, J 7.4, 4.6) and H-18 (δ_H2.06, br t, J 9.4) in the

COSY-45 spectrum, revealing that the vinyl group is positioned

at C-17 and C-28. Observation of a strong NOE between

Me-27 (δ_H1.14, s) and H-18 (δ_H2.06, br t, J 9.4) showed the

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correlated with δ_C110.6 (t, C-29) in the HMQC spectrum, were

found in the H NMR spectrum of 41. The apparent HMBC

cross peaks of the vinyl protons (H-29) for C-30 (δ_C26.0), C-22

(δ_C150.0) and C-21 (δ_C56.4) indicated that an isopropenyl

moiety was linked to C-21. In the HMBC spectrum, Me-28

(δ_H0.97, s) had cross peaks for C-21 and C-18 (δ_C42.6). In

addition, Me-27 (δ_H1.12, s) had a strong HMBC for H-13

(δ_H1.68, m). The clear NOEs of Me-27/H-18 and Me-28/H-21

indicated an α-orientation for H-18 (δ_H1.78, m) and β-orien-

tation for H-21, respectively. These ﬁndings revealed that 41

had a 6/6/6/6/5-fused pentacyclic ring system like 2, but the

methyl group at C-18 of 2 was placed at C-17 for 41. Product 42

also had a 6/6/6/6/5-fused pentacyclic skeleton according to

the detailed NMR analyses, but the stereochemistry at C-21

was opposite to that of 41; H-18 (δ_H1.42, m) was placed in

α-orientation, since clear NOEs of Me-27 (δ_H1.05, s)/H-18 and

H-18/H-21 (δ_H2.15, t, J 9.4) were found, in addition to the

absence of NOE between Me-28 and H-21. Polar compounds

43 and 44 contained a hydroxy group (δ_C73.8 and 72.7, respect-

ively), the position of which was determined to be at C-22 by

the ﬁnding of strong HMBC cross peaks of Me-29 and Me-30

(δ_H1.27, s and 1.25, s, for 43; δ_H1.35, s and 1.21, s, for 44) for

C-22. A strong NOE between Me-28 (δ_H0.96, s) and H-21

(δ_H1.75, m) was observed for 43, but no NOE between them for

44, suggesting β-orientation for H-21 of 43, and α-orientation

for that of 44. The product distribution ratio (%) was deter-

mined by the GC analysis to be as follows: 6.6 : 4.6 : 4.7 : 7.3 :

10.9 : 25.5 : 18.7 : 19.7 for 37, 38, 39, 41, 42, 40, 43, 44, respect-

ively, and 2.0 for the recovered 15. It is noteworthy that all the

tetracyclic skeletons of 37–40 consisted of a 6/6/6/6-fused ring

system (six-membered D-ring), while a dammarane skeleton

(a 6/6/6/5-fused tetracyclic ring system: ﬁve-membered D-ring)

was not constructed, that is believed to be one of the inter-

mediates in the bioconversion of 1 to 2.¹The tetracyclic ring

systems of 37–40 were constructed according to a Markovnikov

closure. The pentacyclic products 41–44 also were produced in

a high yield (total yield, 56.6%). It is remarkable that 15 under-

goes the polycyclization as 15a, but not as 15b (see Scheme 4).

Thus, the cyclization pathway was directional.

Enzymatic reaction of 16 and 17

The conversion from 16 was low (7%) and only one product 45

was found (Fig. 1(D)). Analog 16 (20 mg) was incubated with

the cell-free homogenate (200 cm³) from a 4 L culture of the

cloned E. coli at the optimum catalytic conditions for 16 h. The

quantity of the cell-free homogenates employed was about 6

times higher than that for 15 in order to isolate a suﬃcient

amount of 45 for spectroscopic analyses. The hexane extracts

were subjected to column chromatography over SiO₂by eluting

with hexane, giving pure 45 (yield, 2.3 mg). The presence of

three allyl methyl groups (δ_H1.70, s, Me-29; 1.78, s, Me-28;

1.82, s, Me-30) and two oleﬁnic protons (δ_H5.49, br t, J 6.9, H-

17; 5.39, br t, J 6.8, H-21) suggests that 45 had a tricyclic skel-

eton. The vinyl protons (δ_H4.84, s; 5.10, s: H-27) were also

observed, that had correlations with C-14 (δ_C56.7) and C-12

(δ_C38.7) in the HMBC spectrum, demonstrating that the vinyl

group was arranged as shown in the structure of 45 in Fig. 2. It

is interesting that 45 consisted of a 6/6/6-fused tricyclic ring

system, which was produced according to Markovnikov

closure, but the yield was very low. Analog 17, which has

methyl groups at C-11 and C-14 on the squalene backbone, was

incubated to test whether 17 can be recognized as a substrate.

No detectable amount of the enzymic product was found and

was marginal at best (Fig. 1(E)).

Scheme 4 All pre-chair conformation adopted during the polycycliz-

ation processes of 1, 14 and 15 (top), and the reaction process of 15

leading to the enzymatic products 37–44 (bottom). Two cyclization

pathways (a and b) are possible for 14 and 15, but 1 is a symmetrical

molecule, thus paths a and b lead to the identical all pre-chair

conformation (1a,b). The symbol ᭹ shows the steric hindrance due to

1,3-diaxial interactions between each of methyl groups.

α-orientation of H-18. Thus the structure of 39 was determined

as shown in Fig. 2. The polar compound 40 had a tertiary

alcoholic carbon (δ_C73.4, C-17) and one oleﬁnic proton

(δ_H5.47, br t, J 7.2, H-21), the latter being correlated with the

two allyl methyl signals (δ_H1.84, s, Me-30 and 1.78, s, Me-29) in

the COSY-45 spectrum. Me-28 (δ_H1.15, s) had HMBC corre-

lations for C-17 and C-18 (δ_C48.1). Strong NOEs of Me-28/

H-13 (δ_H1.41, m) and Me-27 (δ_H1.05, s)/H-18 (δ_H1.28, m)

indicated that H-18 was of α-arrangement. Thus, 40 consisted

of a 6/6/6/6-fused tetracyclic ring system having a hydroxy

group. The vinyl protons (δ_H4.84, s; δ_H5.09, s, CH₂-29), which

Discussion

As shown in Scheme 2, the methyl group(s) at the terminal

double bond(s) plays crucial roles in forming a hopane

skeleton⁶(see the cyclization products 6–8 from substrate

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analogs 4 and 5). In addition, the C(10)–Me at the central part

of 1 has a pivotal role in adopting the all-chair conformation

during the polycyclization reaction (path a of 9).⁷Next, we

investigated how the methyl group at C-6 of 1 has an inﬂuence

upon the polycyclization cascade. The C(6)-norsqualene 14

showed a signiﬁcantly high conversion (ca. 98%). Substrate 14

has two isopropylidene moieties at the both terminal sides, thus

the polycyclization reaction can start from both the left (path a,

via intermediate 14a) and right sides (path b via 14b). The

structures of products 34–36 indicated that the polycyclization

reactions of both 14a and 14b occurred with a folding of the all

pre-chair conformation in a similar way as that of 1. The

absence of the methyl group at C-6 (14a) or C-19 (14b) showed

no inﬂuence upon the construction of a hopane skeleton,¹¹in

contrast to the unusual conformation adopted for 9a (Scheme

5). However, the amount of 34 ϩ 35 produced (64%) was about

three times higher than that of 36 (20%), suggesting that 14b

was more preferably recognized as the substrate than 14a and

that a presence of the methyl group at C-6 eﬀectively leads to

the formation of the hopane skeleton. This ﬁnding is very

informative for the insight into the polycyclization mechanism;

the 1,3-diaxial interactions between each of the β-arranged

methyl groups at C-2, C-6 and C-10 are elevated for 1a (1b) or

14b, compared to that for 14a lacking the methyl group at C-6,

but the polycyclization reaction of 1a,b or 14b more eﬃciently

proceeded than that of 14a, suggesting that the cyclase enzyme

has the binding site that can accept the methyl group at C-6. As

an alternative explanation, a Markovnikov preference may

have given rise to the eﬃcient polycyclization reaction, because

the tertiary cation can be formed at C-6 of 14b during the

polycyclization. In the case of substrate 1, 2 is produced in a

signiﬁcantly higher yield, compared to 3 (2 : 3 = 5 : 1), but 14b

gave a nearly equivalent amount of the hydroxylated product 35

relative to that of the deprotonation product 34 (35 : 34 = 1.2 :

1). Now, it is well accepted that the “front water” acts as a

catalytic base to abstract the proton from the (23Z)-Me of 1 to

form 2.^2,4,5aThe methyl group at C-19 of 1 may have a role in

placing the (23Z)-Me at the correct position to facilitate the

introduction of the double bond of 2, but the decreased steric

bulk size at C-19 of 14b led to close proximity of the “front

water” to the ﬁnal hopanyl cation of 14b. An optimal size at

C-19 is a methyl group, whereas a hydrogen atom is small.

Analog 15 also can undergo two cyclization pathways, i.e., path

a (15a) and b (15b). Compounds 15a and 15b have a methyl

group at C-18 and C-7, respectively. Almost full conversion

(98% yield) was found for 15a, but no cyclization for 15b. A

diﬀerence between 15b and 14a is found only at C-7. An intro-

duction of the methyl group at C-7 into 14a completely stopped

the polyclization reaction.

A relatively high conversion

occurred for 14a (20%), but no cyclization product was found

from 15b (Scheme 4). This suggests that a chair conformation

shown in 15b would not have been organized around A- and

B-rings during the polycyclization process due to the repulsive

interaction between the (C)7-Me of 15b and the substrate

recognition site of the cyclase. As for the steric repulsion

between each of methyl groups at C-2, C-6 and C-10, that for

15a is greater than for 15b. However, 15a was eﬃciently cyclized

(98%), while no reaction occurred for 15b. Thus, it can be

concluded from the substrate analogs 14, 15, 4 and 5 that three

methyl groups at C-2, C-6 and C-10, which are arranged in

β-orientation, are critical to the eﬃcient polycyclization

reaction, despite great steric hindrance between each methyl

group involved. Introduction of a methyl group at C-18 of 14b,

i.e., 15a, aﬀorded 6/6/6/6-fused tetracyclic products 37–40 and

the 6/6/6/6/5-fused pentacyclic skeletons 41–44. The abortive

tetracyclic products were produced according to a Markov-

nikov closure (bottom of Scheme 4), the tertiary cation (15c,d)

being formed at C-18. The stereochemistry at C-21 of 42 and 44

was opposite to that of 41 and 43 (natural type), suggesting that

two diﬀerent folding conformations (15c and 15d) are involved

in the polycyclization cascade, as shown in Scheme 4 (bottom).

The deprotonation reactions from 15c and/or 15d gave 37 and

39, respectively. A sequential reaction of 1,2-shifts of hydride

and methyl group in an anti-parallel fashion gave 38. Attack by

water on 15c and/or 15d aﬀorded 40. A further cyclization gave

pentacyclic cations 15e and 15f. The deprotonation reactions

from the cations gave 41 and 42 and attack of water to the

cations gave 43 and 44. The diﬀerence between 15a and 14b

is found only at C-18 position; a large methyl group is

involved in 15a, but a less bulky hydrogen atom in 14b. Only

the pentacyclic skeleton was found from 14b as major products,

but a signiﬁcantly large amount of tetracyclic products

(ca. 41%) was formed from 15a, strongly indicating that the

methyl group at C-18 interrupted the completion of the

polycyclization reaction. Thus, a small hydrogen atom must

be situated at C-18 for the full conversion of 1 into 2. This

interruption would have occurred due to the repulsive inter-

action between C(18)–Me of 15a and the recognition site

nearest to C-18, which is involved in the cyclase enzyme, and

this repulsion also may have led to the improper folding

conformation 15d. The GC analysis (Fig. 2) showed that

15c and 15d were produced in a nearly equivalent amount

[(42ϩ44) : (41ϩ43) = 1 : 1.2]. The experimental results

obtained from 14b and 15a revealed that inappropriate bulk

size around C-18 and C-19 disturbed the normal polycycliz-

ation pathway.

Scheme 5 Cyclization pathways of substrate analogs 9, 16 and 17.

Analog 17 is a symmetrical molecule similarly to 1, thus paths a and b

result in an identical conformation as shown in 17a,b. The symbol ᭹

shows the steric hindrance due to 1,3-diaxial interactions between each

of methyl groups.

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 1 2 – 2 0 2 2

2017

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Analog 16 was converted into 45 (6/6/6-fused tricyclic ring

system) via the folding conformation 16a, but the yield was low

(7%). No detectable amount of the cyclization product via 16b

was found in the incubation mixture. The tricycle 45 was

formed via the tertiary cation 16c according to Markovnikov

preference, but was not via the secondary cation 16d (Scheme

5). When 1 was incubated with a variety of site-directed

mutants, the 6/6/5-fused tricycle 1d only was trapped without

isolation of the 6/6/6-fused tricycle 1c (Scheme 5).^1,12,13More-

over, the trapping experiment of the secondary cation 1c failed

even by using the squalene analogs having a highly nucleo-

philic hydroxy group, and the tertiary cation 1d only was

trapped.^1,12a,b,14These ﬁndings obtained from 1 and 16 may sug-

gest that a stable tertiary cation having a signiﬁcantly longer

lifetime is involved during the polycyclization reaction.¹⁵

Previously, we have reported the cyclization products of 9.⁷The

novel carbocyclic skeleton(s) consisting of a 6/5ϩ5/5ϩ(6) ring

system was constructed with a conformational change from 9a

into 9c (ca. 44%), while hopane skeletons were formed via 9b in

high yields (38%). The diﬀerence between 16a and 9b is found

only at C-14. A small hydrogen atom at C-14 (9b) led to

complete polycyclization, but the introduction of a large methyl

group at C-14 halted the polycyclization at the premature

tricyclic stage (45) and the yield was low (7%). This ﬁnding

suggested that the steric bulk size at C-14 is crucial to guide

further cyclizations; the bulk size of a methyl group at C-14 is

too large. The β-arranged methyl group at C-14 of 16a may

repulsively interact with the 10β-methyl group of 16a and/or

with the substrate recognition site nearest to C-14, giving the

low yield (7%) of the Markovnikov product 45. No cyclization

of 16b also indicated that the bulk size at C-11 must be small;

a methyl group is too large to form a chair conformation

around A/B-rings. Indeed, 9a having a small hydrogen at C-11

underwent the polycyclization reaction in a high yield (44%)

with an unusual conformation being adopted (Scheme 5). The

importance of the accurate size of the attached group at C-11

was further demonstrated by the incubation experiment of

analog 17; no cyclization product was found as shown in

Fig. 1(E). In the all pre-chair conformation, the 1,3-diaxial

repulsive interactions found in 15b and 17 are diminished,

compared with that found in 1a,b, but no cyclization occurred

for 15b and 17. This ﬁnding further indicated that the methyl

groups repulsively interacted with the substrate binding pockets

nearest to C-7 and C-11.

folding conformation around D/E-ring formation. Substitution

with a small hydrogen atom at C-15 (9b) gave the stereo-

chemistry of 21α-H in a signiﬁcantly high yield (9d), which is

opposite to that of 9e given by the normal polycyclization

pathway (9d : 9e = 1.4 : 1). The importance of appropriate steric

bulk size is also true of 14b, which gave a higher production of

the hydroxylated product 35, compared to 1a,b. Incorporation

of a methyl group into C-18 (15a) also halted the polycycliz-

ation reaction at the tetracyclic ring stage. Replacement with a

small hydrogen atom at C-23 led to an abnormal cyclization

product having a six-membered E-ring.^4a,6Replacement of the

methyl group at diﬀerent positions, C-7 (15b, 17a,b) and C-11

(16b), led to no cyclization. An introduction of a methyl group

at C-14 also interrupted the polycyclization. This study veriﬁed

that the methyl positions are important for leading to com-

pletion of the normal polycylization reaction and further

demonstrated that the precise steric bulk size at the methyl

positions of 1 is critical to the correct folding and the strong

binding of the substrate to the squalene cyclase.

Experimental

Analytical methods

NMR spectra were recorded in C₆D₆on a Bruker DMX 600 or

DPX 400 spectrometer, the chemical shifts being relative to the

solvent peak δ_H7.280 and δ_C128.0 ppm as the internal reference

for ¹H and ¹³C NMR spectra, respectively. The NMR spectra of

all the enzymatic products were measured in C₆D₆. Some

synthetic intermediates were measured in CDCl₃. The chemical

shifts in CDCl₃solution were given according to the internal

solvent peaks of δ_H7.26 and δ_C77.0 ppm. The coupling

constants J are given in Hz. GC analyses were done on a

Shimadzu GC-8A chromatograph equipped with a ﬂame

ionization detector (DB-1 capillary column (0.53 mm × 30 m);

injection temperature, 290 ЊC; column temperature, 270 ЊC; N₂

carrier pressure, 1.0 kg cm^Ϫ2). GC-MS spectra were on a JEOL

SX 100 spectrometer under electron impact at 70 eV with a DB-

1 capillary column (0.32 mm × 30 m), the oven temperature

being elevated from 220 to 270 ЊC (3 ЊC min^Ϫ1). HR-EIMS was

performed using a direct inlet system. Speciﬁc rotation values

were measured at 25 ЊC with a Horiba SEPA-300 polarimeter.

Incubation conditions

In conclusion, the substrate analogs used in this study clearly

demonstrated that substrate binding pockets are involved which

accept the methyl groups of 1. The methyl groups at C-2, C-6

and C-10, which are orientated in the β-face, are strongly

captured by the cyclase to aﬀord chair conformations around

the A/B-ring formation, despite great repulsions due to the

occurrence of 1,3-diaxial interactions (see 1a,b of Schemes 4

and 5). The involvement of the thermodynamically stable

Markovnikov cations generated during the polycyclization

process would further overcome the 1,3-diaxial steric repul-

sions. The methyl group at C-2 is indispensable for the cycliz-

ation initiation.^4a,6As shown in Scheme 5, 9a lacking C(10)–Me

was folded in an unusual conformation in the enzyme cavity.

The less bulky hydrogen at C(10) of 9a could not precisely

interact with the binding site of C(10)–Me; in turn, the C(15)–

Me could be strongly captured by the binding pocket intrinsic

to C(10)–Me of 1 through the folding conformation shown in

9c.⁷Thus, the C(15)–Me of 9a could have a β-orientation as

shown in 10 and 11. In addition, a small hydrogen atom at C-6

of 14a also could not correctly interact with the binding pocket

inherent to C(6)–Me of 1, leading to lower production of 14a

compared with that of 14b. The methyl groups at C-15 and

C-19 along with the Z-Me at C-23, which are arranged in α-face

(1a,b), have a crucial role in assembling a half chain of the

squalene molecule ranging from C-15 to C-23 into the

exact position in the reaction cavity, leading to the correct

Standard incubation conditions were performed according to

the published protocols.^16,17The cell-free extract was prepared

as follows. A 1 L culture of E. coli encoding the native SHC

from Alicyclobacillus acidocaldarius was harvested by centri-

fugation and to the collected pellets was added 50 cm³of citrate

buﬀer solution (pH 6.0). Ultrasonication was then carried

out to disrupt the cells. The supernatant was used for the

incubations after removing the cell debris by centrifugation.

1 cm³of the supernatant contains ca. 200 µg of the pure SHC,

which had a speciﬁc activity of ca. 1900 nmol min^Ϫ1mg^Ϫ1for

the substrate 1.^16,17

Preparation of isoprenyl bromides

1-Bromo-3-methylbut-2-ene 18, geranyl bromide 19 and

geranylgeranyl bromide 20 were prepared by treatment of the

corresponding alcohols with PBr₃in THF at 0 ЊC for 30 min.

The reaction mixture was poured into ice-water. The product

was extracted with n-hexane and dried over anhydrous Na₂SO₄

and used without further puriﬁcation. ¹H NMR data (400

MHz, CDCl₃): 18: δ_H1.71 (3H, s), 1.76 (3H, s), 4.00 (2H, d,

J 7.8), 5.50 (br t, J 7.8). 19: δ_H1.60 (3H, s), 1.68 (3H, s), 1.73

(3H, s), 2.1–2.0 (4H, m), 4.01 (2H, d, J 8.4), 5.05 (1H, br t,

J 6.8), 5.53 (1H, t, J 8.4). 20: δ_H1.60 (6H, s), 1.68 (3H, s), 1.73

(3H, s), 2.2–1.95 (14H, m), 4.08 (2H, d, J 8.4), 5.09 (3H, m),

5.53 (1H, t, J 8.4).

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 1 2 – 2 0 2 2

2018

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Synthesis of (E,E,E,E )-2,6,10,14,19,23-hexamethyltetracosa-

2,6,10,14,18,22-hexaene 16

stirred for 30 min. Then, the reaction mixture was poured into

aq. dilute HCl, the ether layer being washed with saturated

Na₂CO₃and dried over anhydrous Na₂SO₄, followed by puriﬁ-

cation with SiO₂column chromatography eluting with a

mixture of hexane–EtOAc (100 : 10) to aﬀord pure 24 in a yield

To a stirred solution of 19 (320 mg, 1.47 mmol) in DMF

(5 cm³), 240 mg of benzenesulﬁnic acid sodium salt (1.46 mmol)

was added under N₂atmosphere and stirred over night. The

reaction mixture was poured into ice-water, extracted with

n-hexane and dried over anhydrous Na₂SO₄. After evaporation

of hexane, the residue was subjected to SiO₂column chromato-

graphy eluting with a mixture of hexane and EtOAc (100 : 10)

to give (3,7-dimethylocta-2,6-diene-1-sulfonyl)benzene 21 (380

mg, 92% yield) in a pure state. NMR data in CDCl₃: δ_H1.31

(3H, s), 1.58 (3H, s), 1.68 (3H, s), 1.99 (3H, s), 3.80 (2H, d,

J 8.0), 5.01 (1H, m), 5.20 (1H, t, J 8.0), 7.53 (2H, t, J 7.2), 7.63

(1H, br t, J 7.2), 7.86 (2H, br d, J 7.8); δ_C17.7 (q), 25.7 (q), 26.2

(t), 39.6 (t), 56.1 (t), 110.3 (d), 123.4 (d), 128.6 (d, 2 × C), 128.9

(d, 2 × C), 132.1 (s), 133.5 (d), 138.7 (s), 146.3 (s). To a solution

of 21 (380 mg, 1.37 mmol) dissolved in 5 cm³of THF and

[(CH₃)₂N]₃PO (4 : 1), 2.0 cm³(mmol) of n-BuLi (1.58 M) was

added at Ϫ20 ЊC to give an orange color, and stirred for 20 min.

The temperature was further lowered to Ϫ78 ЊC and 550 mg

(1.56 mmol) of 20 in THF (1.5 cm³) was added dropwise and

stirred for 1 h. The reaction mixture was poured into ice-water

and extracted with n-hexane and dried over anhydrous Na₂SO₄,

followed by puriﬁcation with SiO₂column chromatography, to

aﬀord the pure sulfone derivative 22 (290 mg, yield 38.5%).

NMR data of 22 (400 MHz, CDCl₃): δ_H1.19 (3H, s), 1.56 (3H,

s), 1.58 (6H, s), 1.59 (3H, s), 1.61 (3H, s), 1.68 (6H, s), 2.0–1.9

(16H, m), 2.34 (1H, m), 2.88 (1H, m), 3.73 (1H, dt, J 3.2, 10.4),

5.1–4.9 (6H, m), 7.50 (2H, t, J 8.0), 7.60 (1H, d, J 7.6), 7.84 (2H,

d, J 7.6); δ_C15.92 (q), 15.96 (q), 16.34 (q), 16.47 (q), 17.64 (q,

2 × C), 25.65 (2, 2 × C), 26.23 (t), 26.42 (t), 26.59 (t), 26.64 (t),

26.73 (t), 39.69 (t, 3 × C), 39.73 (t), 64.82 (d), 117.0 (d), 118.6

(d), 123.6 (d), 123.8 (d), 124.1 (d), 124.3 (d), 128.6 (d, 2 × C),

129.1 (d, 2 × C), 131.2 (s), 131.9 (s), 133.3 (d), 134.9 (s), 135.2

(s), 138.1 (s), 138.6 (s), 145.1 (s). The deprotection reaction of

the phenylsulfone group was done with LiBEt₃H and a catalytic

amount (3 mg) of PdCl₂(dppp) in Et₂O. The ethereal solution

of the sulfone (28.7 mg/5 cm³) was cooled at 0 ЊC. To the solu-

tion, 2.0 cm³of 1.0 M LiBEt₃H was added, and stirred for 1 h

and poured into ice-water, and the hexane-extract was puriﬁed

with SiO₂column chromatography eluting with n-hexane to

give pure 16 (19.0 mg yield, 89%). NMR data of 16 (400 MHz,

C₆D₆): δ_H1.69 (6H, s), 1.73 (12H, s), 1.80 (6H, s), ∼2.3 (20H,

m), ∼5.4 (6H, m); δ_C16.1 (q), 17.7 (q), 25.8 (q), 27.1 (t), 27.2 (t),

28.7 (t), 30.4 (q), 40.2 (t), 124.8 (d), 124.8 (d), 124.9 (d), 131.1

(s), 134.9 (s), 135.1 (s). EIMS: m/z 69 (100%), 91 (65), 137 (16),

410 (M^ϩ, 3).

of 84.5 mg (80 %). H NMR data of 24 (400 MHz, CDCl₃):

δ_H1.18 (3H, d, J 6.0), 1.60 (6H, s), 1.61 (3H, s), 1.67 (3H, s),

∼2.0 (16H, m), 3.77 (1H, m), ∼5.12 (4H, m). EIMS: m/z 69

(100), 81 (47), 109 (64), 137 (12), 333 (M^ϩ, 3). To obtain 25 from

24, Collin’s reagent was prepared. To a solution of pyridine (1.5

g, 19.0 mmol) in CH₂Cl₂(5 cm³), 324 mg of CrO₃(3.2 mmol)

was added and stirred for 2 h, then the reaction ﬂask was cooled

to 0 ЊC. A solution of 24 (90 mg, 0.27 mmol) in CH₂Cl₂(3 cm³)

was slowly added and stirred for 12 h, and then anhydrous

MgSO₄was added. The resultant precipitates were ﬁltered oﬀ,

followed by puriﬁcation with SiO₂column chromatography by

eluting with a mixed solvent of hexane and EtOAc (100 : 5) to

give 25 (yield, 60.5 mg, 68%). NMR data of 25 (400 MHz,

CDCl₃): δ_H1.59 (12H, s), 5.10 (4H, m), 1.67 (3H, s), ∼2.0 (12H,

m), 2.12 (3H, s), 2.23 (2H, t, J 7.6), 2.50 (2H, t, J 7.6); δ_C, 15.93

(q), 15.98 (q), 16.00 (q), 17.61 (q), 25.63 (q), 25.58 (t), 26.70 (t),

28.04 (t), 28.16 (t), 29.79 (q), 33.54 (t), 39.67 (2 × C, t), 42.37 (t),

124.0 (d), 124.2 (d), 124.3 (d), 124.9 (d), 131.2 (s), 133.5 (s),

134.8 (s), 135.2 (s). 208.7 (s, C᎐O). EIMS: m/z 69 (100%), 81

᎐

(62), 125 (41), 330 (M^ϩ, 3). NaH (24 mg, 1.0 mmol), obtained

after washing with hexane, was added to the THF solution of

(EtO)₂POCH₂CO₂Et (336 mg, 1.5 mmol/5 cm³) and stirred for

30 min under N₂atmosphere. To the solution, 25 (100 mg, 0.3

mmol) in 5 cm³of THF was added and stirred for 12 h. The

reaction mixture was poured into ice-water and extracted with

hexane. SiO₂column chromatography eluting with a mixture of

hexane and EtOAc (100 : 2) gave pure 26a in a yield of 88.3 mg

(74%). ¹H NMR data of 26a (400 MHz, CDCl₃): δ_H1.27 (3H, t,

J 7.2), 1.57 (3H, s), 1.60 (12H, s), 1.68 (3H, s), ∼2.0 (16H, m),

4.14 (2H, q, J 7.2), 5.11 (4H, m), 5.65 (1H, s). EIMS: m/z 69

(100%), 81 (85), 128 (51), 400 (M^ϩ, 19). The ester 26a was

reduced with DIBAL-H as follows. To the solution of 26a

(100 mg, 0.25 mmol) dissolved in Et₂O (5 cm³), 1.5 cm³of

(iso-Bu)₂AlH (0.95 M in n-hexane) was slowly added and stirred

for 1 h. The reaction mixture was poured into aq. EtOAc,

stirred for 2 h and then aq. saturated NH₄Cl was added, the

resultant precipitated salts being ﬁltered oﬀ. After evaporation

of the organic layer, the residues were subjected to SiO₂column

chromatography eluting with 20% EtOAc–hexane to yield 27a

(70 mg, 78%). ¹H NMR data of 27a (400 MHz, CDCl₃): δ_H1.60

(12H, s), 1.67 (6H, s), ∼1.98 (8H, m), ∼2.06 (8H, m), 4.12 (2H,

J 6.8), 5.11 (4H, m), 5.40 (1H, t, J 6.4). EIMS m/z 69 (100%), 81

(82), 93 (40), 135 (29), 358 (M^ϩ, 3). 3-Methylbut-2-en-1-ol and

27a were treated with PBr₃to prepare the corresponding brom-

ides 18 and 28, respectively. 18 was converted into 29 according

Synthesis of (E,E,E,E )-2,6,10,15,18,23-hexamethyltetracosa-

2,6,10,14,18,22-hexaene 15

to the method described in the preparation of 21. H NMR

The synthetic intermediate 24 was prepared as follows. Com-

pound 1 (1.01 g, 2.46 mmol) dissolved in 25 cm³of CH₂Cl₂was

treated with m-CPBA (629 mg, 3640 mmol) at 0 ЊC for 2 h to

give a mixture of 2,3-, 6,7 and 10,11-oxidosqualenes. The

reaction mixture was washed with 20% aq. NaHCO₃and brine,

and the organic layer was dried over anhydrous Na₂SO₄(yield,

960 mg). A mixture of the products (11.88 g, prepared by

repeated experiments) was treated with H₅IO₆(9.49 g), followed

by puriﬁcation with a reverse-phased open column (C₁₈) by

stepwise elution using aq. CH₃CN (75% CH₃CN to 100%

CH₃CN), giving the pure C₂₂-aldehyde 23 (3.1 g). ¹H NMR data

of 23 (400 MHz, CDCl₃): δ_H1.59 (15H, s), 1.60 (3H, s), 1.65

(3H, s), ∼2.0 (12H, m), 2.30 (2H, t, J 6.8), 2.11 (2H, t, J 7.2),

2.23 (6H, m), 2.30 (2H, t, J 8.0), 2.50 (2H, t, J 8.0), 5.10 (4H,

m), 9.74 (1H, s). EIMS: m/z 69 (100), 81 (100), 316 (M^ϩ, 20). To

a suspension of Mg metal (19.2 mg, 0.8 mmol) in 5 cm³of Et₂O,

the solution of CH₃I (454 mg, 3.4 mmol) in Et₂O (5 cm³)

was added and reﬂuxed for 30 min. To the Grignard reagent, a

solution of 23 (100 mg) in Et₂O (5 cm³) was slowly added and

data of 29 (400 MHz, CDCl₃); δ_H1.27 (3H, s), 1.66 (3H, s), 3.74

(2H, br d, J 7.6), 5.14 (br t, J 6.8), 7.50 (2H, t, J 7.2), 7.60 (1H,

J 7.2), 7.82 (d, J 7.6). A solution of 29 (100 mg, 0.476 mmol),

dissolved in 10 cm³of the mixed solvent of THF and

[(CH₃)₂N]₃PO (4 : 1), was cooled at Ϫ20 ЊC and then 2.0 cm³of

n-BuLi (1.58 M in n-hexane) was added, the color changing to

orange, and stirred for 20 min. The temperature was further

lowered to Ϫ78 ЊC. To the cooled solution, 28a (75 mg, 0.178

mmol) in THF (5 cm³) was added and stirred for 1 h. The

reaction mixture was poured into ice-water, extracted with

hexane, and dried over anhydrous Na₂SO₄. Puriﬁcation was

done with SiO₂column chromatography eluting with 10%

EtOAc–hexane, giving pure 30a in a yield of 52.6 mg (79%).

NMR data of 30a (400 MHz, CDCl₃): δ_H1.16 (3H, s), 1.55 (3H,

s), 1.58 (12H, s), 1.65 (3H, s), 1.66 (3H, s), ∼2.0 (16H, m), 2.33

(1H, m), 2.83 (1H, m), 3.68 (1H, ddd, J 10.7, 10.7, 3.6), 4.95

(2H, t, J 7.2), 5.10 (4H, m), 7.50 (2H, t, J 7.6), 7.60 (1H, t,

J 7.6), 7.82 (2H, d J 7.2). The phenylsulfone group was removed

by using LiBEt₃H and a catalytic amount of PdCl₂(dppp) in

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 1 2 – 2 0 2 2

2019

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Et₂O. Compound 30a (50 mg, 0.09 mmol) was dissolved in

5 cm³of diethyl ether and was cooled at 0 ЊC. To the solution,

1.0 cm³of 1.0 M LiBEt₃H was added and stirred for 30 min and

poured into ice-water, and then extracted with hexane and

puriﬁed with SiO₂column chromatography by eluting with

n-hexane to give pure 15 (28.1 mg yield, 75%). NMR data of 15

(400 MHz, C₆D₆): δ_H1.69 (6H, s), 1.73 (12H, s), 1.80 (6H, s),

∼2.3 (20H, m), ∼5.4 (6H, m); δ_C16.1 (q), 16.2 (q), 17.7 (q), 17.8

(q), 25.8 (q), 27.1 (t), 27.2 (t), 28.7 (t), 28.8 (t), 38.9 (t), 40.2 (t),

124.8 (d, 2 × C), 124.9 (d), 125.0 (d), 131.1 (s), 131.3 (s), 134.9

(s), 135.1 (s), 135.3 (s). EIMS: m/z 69 (100%), 81 (78), 95 (31),

137 (22), 410 (M^ϩ, 4).

eluting with hexane–EtOAc (100 : 20) (yield, 380 mg, 76%).

EIMS m/z of 33: 69 (100), 81 (23), 93 (28), 137 (12), 180 (M^ϩ,

7). 33 thus obtained was subjected to the Wittig reaction with

Ph P᎐C(CH )CO Et¹⁸under reﬂux in benzene to obtain 2,7,11-

᎐

trimethyldodeca-2,6,10-trienoic acid ethyl ester; EIMS: m/z 69

(100%), 81 (43), 128 (35), 264 (M^ϩ, 13). From 1.5 g of 33, 1.4 g

of the ethyl ester was obtained (yield, 64%). The ester was then

reduced with LAH to the corresponding alcohol (yield, 68%),

EIMS: m/z 69 (100%), 81 (40), 95(27), 137 (30), 204 (4), 222

(M^ϩ, 3). The alcohol (400 mg) was then treated with PBr₃to

give the corresponding bromide, which was then subjected to

allyl coupling reaction¹⁰to give the desired substrate 17 as

follows: to the stirred solution of pyrrolidine (250 mg, 3.5

mmol) in dry Et₂O, was slowly added 2.1 cm³of n-BuLi (1.58 M

in hexane) at 0 ЊC and the solution allowed to stand for 20 min

under N₂atmosphere. Powdered CuI (250 mg, 1.3 mmol) was

added in small portions, the color changing to reddish brown.

After 20 min, a solution of 100 mg of the C₁₅-bromide (0.35

mmol) in Et₂O (15 cm³) was slowly added and stirred for 90

min. The reaction mixture was poured into ice-water and the

product was extracted with hexane and dried over anhydrous

Na₂SO₄. The desired product 17 was obtained by purifying with

5% AgNO₃–SiO₂column chromatography. The coupling yield

was small, but 6.8 mg was obtained from 100 mg of the C₁₅-

bromide. Repeated coupling reaction experiments allowed us to

obtain 90 mg of 17. NMR data of 17 (400 MHz, C₆D₆): δ_H5.45

(2H, br t, J 7.1), 5.42 (2H, br t, J 7.1), 5.37 (2H, br t, J 7.1),

2.35–2.20 (20H, m), 1.80 (6H, s), 1.74 (6H, s), 1.73 (6H, s), 1.69

(6H, s); δ_C134.93 (s, 2 × C), 134.94 (s, 2 × C), 131.1 (s, 2 × C),

124.93 (d, 2 × C), 124.86 (d, 2 × C), 124.81 (d, 2 × C), 40.2 (t, 4

× C), 28.7 (t, 2 × C), 27.2 (t, 2 × C), 27.1 (t, 2 × C), 25.8 (q, 2 ×

C), 17.7 (q, 2 × C), 16.15 (q, 2 × C), 16.1 (q, 2 × C). EIMS: m/z

69 (100%), 81 (78), 95 (27), 137 (23), 410 (M^ϩ, 4).

Preparation of (E,E,E,E )-2,6,10,15,23-pentamethyltetracosa-

2,6,10,14,18,22-hexaene 14

The aldehyde 23 was subjected to a Wadsworth–Emmons

reaction with (EtO)₂POCH₂CO₂Et to obtain the desired ester

26b. The preparation protocols from 26b to 14 were essentially

the same as those from 26a to 15, which was described above.

¹H NMR data of 26b (400 MHz, CDCl₃): δ_H1.27 (3H, t, J 7.2),

1.57 (12H, s, 4 × Me), 1.65 (3H, 1 × Me, s), 2.10–1.94 (16H, m),

2.28 (2H, dd, J 7.2), 4.16 (2H, q, J 7.2), 5.09 (4H, m), 5.79 (1H,

d, J 15.5), 6.93 (1H, m). EIMS: m/z 69 (100%), 81 (52), 107 (32),

386 (M^ϩ, 13). ¹H NMR data of 27b (400 MHz, CDCl₃); δ_H1.58

(12H, s), 1.73 (3H, s), ∼2.0 (16H, m), 4.06 (2H, d, J 4.4), 5.09

(4H, m), 5.64 (2H, m). EIMS: m/z 69 (100%), 81 (100), 344 (M^ϩ,

3). NMR data of 14 (400 MHz, CDCl₃): δ_H1.58 (15H, s, 5 ×

Me), 1.66 (6H, s, 2 × Me), 2.10 (20H, m), 5.09 (5H, m), 5.34

(2H, br s); δ_C15.99 (q), 16.03 (q, 2 × Me), 17.68 (q), 17.73 (q),

25.7 (q, 2 × C), 26.6 (t), 26.7 (t), 28.2 (t, 2 × C), 135.1 (s), 31.3

(t), 31.6 (t), 32.8 (t), 39.72 (t), 39.73 (t), 39.78 (t), 124.17 (d),

124.25 (d), 124.27 (d), 124.37 (d), 124.38 (d), 129.9 (d), 130.2

(d), 131.2 (s), 131.5 (s), 134.8 (s), 134.9 (s). EIMS: m/z 69

(100%), 81 (80), 327 (6), 353 (3), 396 (M^ϩ, 7).

Spectroscopic data of products 34–45

Product 34 (solid). 600 MHz in C₆D₆: δ_H0.89 (2H, m, H-1,

H-5), 0.99 (3H, s, Me-24), 1.01 (3H, s, Me-25), 1.03 (3H, s,

Me-23), 1.09 (3H, s, Me-27), 1.11 (m, H-19), 1.20 (m, H-17),

1.29 (m, H-3), 1.34 (m, H-12), 1.35 (m, H-16), 1.38 (m, H-13),

1.44 (m, H-15), 1.49 (m, H-2), 1.50 (2H, m, H-3, H-15), 1.52

(2H, m, H-9, H-10), 1.54 (2H, m, H-7), 1.59 (m, H-6), 1.60 (m,

H-18), 1.62 (m, H-12), 1.64 (m, H-6), 1.71 (m, H-2), 1.74 (m,

H-19), 2.04 (m, H-20), 2.70 (m, H-21), 4.85 (s, H-28), 5.03 (s,

H-28); δ_C15.15 (C-27), 16.13 (C-26), 16.45 (C-25), 18.99 (C-2),

19.07 (C-6), 21.29 (C-12), 21.79 (C-24), 24.44 (C-29), 24.54

(C-11), 27.56 (C-16), 29.62 (C-20), 29.98 (C-19), 32.40 (C-15),

33.44 (C-4), 33.57 (C-23), 34.63 (C-7), 37.81 (C-10), 40.64

(C-1), 40.93 (C-8), 42.09 (C-14), 42.32 (C-3), 42.48 (C-18),

44.87 (C-13), 48.25 (C-21), 50.86 (C-17), 51.30 (C-9), 56.79 (C-

5), 110.70 (C-28), 149.42 (C-22), Assignments of C-2 and C-6

are exchangeable. EIMS: m/z 191 (100%), 353 (25), 381 (18),

396 (M^ϩ, 63). HREIMS: m/z found 396.3713 (M^ϩ, C₂₉H₄₈

requires 396.3756). [α]_D²⁵(C₆H₆) 7.24 (c 0.12).

Synthesis of (E,E,E,E )-2,6,11,14,19,23-hexamethyltetracosa-

2,6,10,14,18,22-hexaene 17

To a solution of malonic dimethyl ester (13.56 g, 0.1 mol) in

EtOH (150 cm³), 64.1 cm³of 1.5 M NaOEt (96 mmol) was

added under N₂atmosphere and the reaction temperature was

cooled to Ϫ20 ЊC. 19 (13.92 g, 64 mmol), dissolved in 50 cm³of

EtOH, was slowly added and stirred for 1 h. The reaction

mixture was poured into ice-water, extracted with hexane and

dried over anhydrous Na₂SO₄. This crude material was stirred

overnight in 1 M KOH/MeOH. The solution was diluted with

water and acidiﬁed with 4 M aq. HCl and extracted with EtOAc

(× 4), and dried over anhydrous Na₂SO₄. After evaporation, the

residues were heated at 130 ЊC to undergo decarboxylation,

followed by the methylation with dry 5% HCl–MeOH under

reﬂux. Puriﬁcation was done with SiO₂column chromato-

graphy (hexane–EtOAc 100 : 2) to give pure 31 (yield 10 g). ¹H

NMR data of 31 (400 MHz, CDCl₃): δ_H1.57 (3H, s), 1.59 (3H,

s), 1.65 (3H, s), 1.95 (2H, br t, J 6.8), 2.02 (2H, br t, J 6.8), 2.33

(2H, br s), 3.64 (3H, s), 5.05 (2H, m). EIMS: m/z 69 (100), 95

(58), 107 (41), 135 (23), 210 (M^ϩ, 32). To a solution of 31 (5.20

g) in THF (150 cm³), powdered LiAlH₄was added in a small

portion at 0 ЊC, the reaction being monitored by TLC, and the

reaction mixture was poured into aq. EtOAc and stirred over-

Product 35 (solid). 600 MHz in C₆D₆: δ_H0.90 (2H, m, H-1, br

d, 9.4, 1.7 Hz, H-5), 0.97, (m, H-19), 0.99 (3H, s, Me-24), 1.01

(3H, s, Me-25), 1.04 (3H, s, Me-23), 1.11 (3H, s, Me-26), 1.14

(3H, s, Me-27), 1.22 (4H, m, H-17, s, Me-29), 1.25 (3H, s,

Me-29), 1.26 (m, H-13), 1.28 (m, H-3), 1.32 (m, H-11), 1.33 (m,

H-12), 1.45 (2H, m, H-15), 1.48 (2H, m, H-18, H-20), 1.49 (2H,

m, H-2), 1.50 (m, H-3), 1.51 (m, H-9), 1.55 (2H, m, H-7), 1.63

(2H, m, H-6), 1.64 (m, H-11), 1.74 (m, H-1), 1.75 (m, H-12),

1.84 (m, H-16), 1.89 (m, H-19), 1.90 (m, H-20), 1.95 (m, H-16),

2.10 (m, H-21); δ_C15.16 (C-27), 16.07 (C-26), 16.62 (C-25),

18.99 (C-2), 19.07 (C-6), 21.32 (C-11), 21.80 (C-24), 25.56

(C-16), 27.53 (2 × C, C-12, C-20), 27.94 (C-28), 29.94 (C-29),

30.98 (C-19), 33.28 (C-15), 33.45 (C-4), 33.58 (C-23), 34.61

(C-7), 37.82 (C-10), 40.66 (C-1), 40.86 (C-8), 41.61 (C-14),

42.33 (C-3), 44.33 (C-18), 45.51 (C-13), 50.12 (C-17), 50.97

night. Filtration gave almost pure alcohol 32 (yield 4.4 g). H

NMR data of 31 (400 MHz, C₆D₆): δ_H1.68 (6H, s), 1.79 (3H, s),

∼2.2 (6H, m), 3.99 (2H, br s), 5.34 (2H, br m), 5.65 (1H, m).

EIMS m/z 57 (100), 86 (30), 95 (16), 139 (7), 182 (M^ϩ, 1). Next,

the alcohol 32 was subjected to the oxidation reaction. To a

stirred suspension of pyridinium chrorochromate (1.2 g, 0.54

mmol) in CH₂Cl₂containing Celite and anhydrous NaOAc (300

mg each), a solution of 32 (500 mg) in CH₂Cl₂(5 cm³) was

added. After further stirring for 30 min, the reaction mixture

was passed through a short SiO₂column to remove the salts by

O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 0 1 2 – 2 0 2 2

2020

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(C-21), 51.37 (C-9), 56.84 (C-5), 73.91 (C-22), Assignments of

C-2 and C-6 are exchangeable. EIMS: m/z 191 (100%), 356 (44),

396 (33), 414 (M^ϩ, 22). HREIMS: m/z found 414.3848 (M^ϩ,

C₂₉H₅₀O requires 414.3862). [α]_D²⁵(C₆H₆) ϩ10.57 (c 0.11).

(H, m, H-2), 1.61 (m, H-11), 1.63 (m, H-13), 1.70 (m, H-19),

1.71 (m, H-15), 1.72 (m, H-1), 1.75 (3H, s, Me-30), 1.84 (3H, s,

Me-29), 1.86 (m, H-19), 1.92 (m, H-12), 2.07 (m, H-18), 2.30

(m, H-20), 2.32 (2H, dd, J7.4, 4.6, H-16), 2.41 (m, H-20), 4.92

(s, H-28), 5.02 (s, H-28), 5.43 (t, J 6.7 Hz, H-21); δ_C15.59

(C-27), 16.01 (C-26), 16.42 (C-25), 17.83 (C-30), 18.92 (C-6),

19.05 (C-2), 21.27 (C-11), 25.55 (C-20), 25.87 (C-29), 26.66

(C-12), 29.16 (C-19), 33.26 (C-16), 33.41 (C-4), 33.57 (C-23),

34.08 (C-15), 34.38 (C-7), 37.67 (C-10), 40.59 (C-1), 41.30

(C-8), 41.87 (C-14), 42.36 (C-3), 43.10 (C-18), 43.17 (C-13),

50.87 (C-9), 56.81 (C-5), 105.51 (C-28), 125.79 (C-21), 130.93

(C-22), 152.12 (C-17). Assignments of C-2 and C-6 and those

of C-13 and C-18 are exchangeable. EIMS: m/z 69 (100%), 191

(71), 231 (70), 395 (74), 410 (M^ϩ, 58). HREIMS: m/z found

Product 36 (solid). 600 MHz in C₆D₆: δ_H0.78 (2H, m, H-1,