Natural product-derived building blocks for combinatorial synthesis. Part 1. Fragmentation of natural products from myxobacteria

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Natural product-derived building blocks for combinatorial

synthesis. Part 1. Fragmentation of natural products from

myxobacteria

1

Jutta Niggemann,*^aKatrin Michaelis,^bRonald Frank,^bNorbert Zander^band Gerhard Höﬂe^a

^aAbteilung Naturstoﬀchemie, Gesellschaft für Biotechnologische Forschung mbH,

Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail: jni@gbf.de

^bAG Molekulare Erkennung, Gesellschaft für Biotechnologische Forschung mbH,

Mascheroder Weg 1, D-38124 Braunschweig, Germany

Received (in Cambridge, UK) 17th July 2002, Accepted 6th September 2002

First published as an Advance Article on the web 17th October 2002

Novel and unique chiral building blocks of high structural diversity were obtained by selective chemical

fragmentation of natural products from myxobacteria. Subsequent modiﬁcation reactions provided primary

alcohol and carboxylic acid derivatives, which are suitable for the construction of combinatorial chemical

libraries. The single SPOT synthesis of a hybrid structure on a polypropylene membrane was employed to

demonstrate the chemical recombination of such rare building blocks on a micro-scale.

investigated in order to increase diversity in the coupling chem-

istry for library synthesis. The SPOT technique^33,34allows the

simultaneous synthesis of large numbers of compounds on a

nanomolar scale and is therefore especially suited for library

synthesis with valuable and rare building blocks. Our strategy

for the preparation of non-target directed combinatorial librar-

ies is demonstrated by the solid-phase synthesis of a hybrid

structure containing two natural product-derived fragments.

Introduction

Natural products continue to play an important role in drug

discovery,^1–4especially in the ﬁeld of anti-tumor and anti-

infective agents. Recently, there has been an increasing interest

in the synthesis of natural product-based combinatorial

libraries^5–8with high structural diversity for the search of new

lead structures and for the identiﬁcation of new targets emer-

ging from the growing eﬀorts in genomics and proteomics.^9,10

At present, two main strategies are applied for the combin-

atorial synthesis of non-oligomeric natural product-based

libraries. The target-oriented library synthesis is based on the

Results and discussion

systematic structural modiﬁcation of

a

natural product

Reductive ring opening of the macrolide soraphen A (1) with

LiAlH₄in THF³⁵and subsequent silylation of the 1,17-diol

with tert-butyldimethylsilyl chloride (TBSCl)–imidazole gave

access to the known tert-butyldimethylsilyl (TBS)-protected

intermediate 7.³⁶Selective cleavage of the ∆^9,10double bond by

ozonolysis, followed by reduction with NaBH₄aﬀorded the

primary alcohol fragments 8 (66%) and 9 (57%) (Scheme 2). The

former was oxidised with pyridinium dichromate (PDC) in

DMF to the corresponding carboxylic acid derivative 10,

isolated as the triethylammonium salt in 61% yield.

skeleton for the optimisation of existing leads (focused libraries)

by either semisynthetic or, with the possibility of introducing

broader structural variations, total synthesis approaches.^5–7,11

Diversity-oriented approaches on the other hand utilise either

common structural motifs of natural products that are capable

of interacting with a wide range of biological targets (‘privi-

ledged structures’) for the synthesis of structural complex and

diverse small-molecule libraries^12–14or multifunctional scaf-

folds (e.g. steroids or carbohydrates) for generating diversity by

introducing variable side chains.^15–17

The 5-O-methylated derivative 12 was obtained in an analo-

gous reaction sequence by ozonolysis of the TBS-protected

5-O-methyl compound 11, which was derived from soraphen A

by 3,5-di-O-methylation with MeI and NaH in HMPA, acid

catalysed removal of the C-3-OMe group and subsequent

reductive cleavage of the lactone bond in 47% overall yield.

Reaction of soraphen A with H₂SO₄in MeOH provided, in a

complex reaction sequence, the γ-lactone 13³⁵(Scheme 3) in

81% yield. When the reaction was performed in CD₃OD an

exchange of the 3-OMe and 17-OMe groups was found. We

assume that the reaction takes places by a concerted mechanism

with elimination of the hydroxy group at C-5 and cleavage of

the macrocyclic lactone with inversion of the stereocenter at

C-17 (no H/D-exchange was found when 13 was reacted with

H₂SO₄in CD₃OD). The structure elucidation was performed by

¹H–¹H COSY, ¹H–¹H TOCSY, and ¹H–¹³C HMBC experiments.

The illustrated stereochemistry at C-3 and C-4 was derived

from NOE diﬀerence NMR spectra and NOE correlations in

the ¹H–¹H ROESY spectrum of compound 15. The 3-OMe and

4-OMe signals show signiﬁcant diﬀerences in their chemical

shifts (3.48 and 3.39 ppm, respectively). NOE correlations were

Here we report on a novel strategy for the construction of

diversity-oriented natural product-based libraries by combin-

atorial chemical synthesis of hybrid structures via recombin-

ation of complex fragments of natural products. The synthesis

of a unique collection of structural diverse chiral building

blocks by chemical fragmentation of a range of natural prod-

ucts from myxobacteria is described. Myxobacteria are a rich

source of bioactive secondary metabolites,¹⁸and during the

past years we isolated highly diverse natural products, such as

the antifungal macrolide soraphen A (1),¹⁹the β-methoxy-

acrylate fungicide myxothiazol A (2a),^20–22the macrolide-

polyether antibiotic sorangicin A (3),^23–25the anticancer drug

candidate epothilone A (4),^26–28the antifungal compound

ambruticin S (5),^29,30or the cytostatic macrolide apicularen A

(6)^31,32(Scheme 1). So far, we have focused on cleavage reactions

that result in most easily accessible and stable primary alcohol

derivatives. Additional functional groups were selectively pro-

tected to provide chiral building blocks suitable for the solid-

phase synthesis of single compound combinatorial libraries.

Moreover, the conversion to carboxylic acid fragments was

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This journal is © The Royal Society of Chemistry 2002

DOI: 10.1039/b206953a

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Scheme 1 Natural products from myxobacteria.

found between 2-Me and 3-OMe, 5-H_band 4-OMe, and 6-Me

and 5-H_b.

of the primary silyl-group with 0.2 equivalents of camphor-

sulfonic acid⁴³(CSA) in MeOH–CH₂Cl₂provided 21 in 83%

yield.

As ozonolysis of γ-lactone 13 gave after reductive work-up

compound 14 as a single product, the ∆^9,10double bond was

ﬁnally dihydroxylated by catalytic OsO₄oxidation in the

presence of 4-methylmorpholine N-oxide (NMO)³⁷and the

1,2-glycol was cleaved with NaIO₄. Subsequent reduction with

NaBH₄aﬀorded both alcohol fragments 14 and 15 in 72% and

74% yields, respectively. Oxidation of 15 with PDC provided

after column chromatography the carboxylic acid 17 (56%).

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-catalysed oxid-

ation^38,39of the primary alcohol 14 in the presence of sodium

chlorite (NaClO₂) and NaOCl gave on the other hand in

comparable yields the carboxylic acid 16 (60%).

Oxidation of the primary alcohol 21 with TEMPO–KBr–

NaOCl provided the carboxylic acid 22 (51%). DIBAL-

H-reduction of the TBS-protected ester 23 which was obtained

in 82% yield from 19 also gave access to the primary alcohol 24

(71%).

Acetylation of sorangicin A (3) with acetic anhydride in

pyridine provided the corresponding 21,22,25-tri-O-acetate⁴⁴

which was cleaved by ozonolysis followed by reaction with

Me₂S and subsequent reduction with borohydride ion exchange

resin⁴⁵(Scheme 5). The substituted tetrahydropyran derivative

25 and the bicyclic ether 26 were isolated by column chrom-

atography in 79% and 84% yields, respectively, after simple

removal of the polymer-supported reagent by ﬁltration. Select-

ive protection of the diol 25 was then achieved by deacetylation

with NaOMe in MeOH, introduction of TBS-protecting

groups to provide the fully silylated compound 27 (58% overall

yield), and subsequent selective deprotection with CSA in

MeOH–CH₂Cl₂to aﬀord the primary alcohol 28 (41%) and the

diol 29 (44%). Selective silylation of 29 with TBSCl–Et₃N–

DMAP in anhydrous CH₂Cl₂provided on the other hand the

primary alcohol 30 (38%). Both 28 and 30 contain traces of

the other regioisomer and were ﬁnally puriﬁed by HPLC.

TBS-protection of diol 26 gave the TBS-protected bicyclic

ether 31 in 57% yield which upon reaction with 0.2 equivalents

of CSA in CH₂Cl₂–MeOH furnished in 42% yield a 1 : 1

Myxothiazol Z (2b),^40,41the ester-analog and co-metabolite

of myxothiazol A (2a), was synthesised following established

methods from myxothiazol A by conversion of the amide into

an ester group.⁴²OsO₄-oxidation without aﬀecting the ∆^2,3

double bond of the β-methoxy acrylate followed by diol cleav-

age with NaIO₄and subsequent NaBH₄reduction provided bis-

thiazole 18 (78%) as racemate and the β-methoxyacryloyl ester

19 (67%) (Scheme 4). The racemic bis-thiazole 18 is formed by

cleavage of the ∆^15,16double bond as a result of a rearrange-

ment reaction during oxidation with OsO₄which is in accord-

ance with the results obtained by ozone-degradation followed

by an oxidative work-up of myxothiazol A.²¹

Silylation of diol 18 with TBSCl–imidazole furnished in

85% yield bis-silyl ether 20. Subsequent selective deprotection

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

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Scheme 4 Reagents: a) 1. OsO₄, NMO, 2. NaIO₄, 3. NaBH₄;

b) TBSCl, imidazole; c) CSA; d) TEMPO, KBr, NaOCl,; e) DIBAL-H.

Scheme 2 Reagents: a) 1. LiAlH₄, 2. TBSCl, imidazole; b) 1. O₃, 2.

NaBH₄; c) 1. PDC, 2. Et₃N; d) 1. MeI, NaH, HMPA, 2. HCl, 3. LiAlH₄,

4. TBSCl, imidazole; e) 1. O₃, 2. NaBH₄.

Scheme 5 Reagents: a) 1. Ac₂O, pyridine, 2. O₃, 3. Me₂S, 4. BH₄^Ϫion

exchange resin; b) 1. NaOMe, MeOH, 2. TBSCl, imidazole; c) CSA;

d) TBSCl, Et₃N, DMAP; e) TBSOTf, Et₃N; f ) CSA.

reduction with BH₄^Ϫion exchange resin the fragments 37 (48%)

and 38 (55%), isolated by preparative HPLC. Here, the use of

polymer-supported BH₄is particularly advantageous as it

makes the isolation of polar compounds like 37 feasible by

avoiding an aqueous work-up. Moreover, probably due to steric

hindrance, no reduction of the carbonyl group at C-3 of com-

Scheme 3 Reagents: a) H₂SO₄, MeOH; b) 1. OsO₄, NMO, 2. NaIO₄, 3.

NaBH₄; c) 1. TEMPO, NaOCl, NaClO₂, 2-methylbut-2-ene, 2. Et₃N;

d) 1. PDC, 2. Et₃N.

Ϫ

mixture of the primary alcohols 32 and 33. Separation of the

regioisomers was ﬁnally achieved by column chromatography.

Treatment of epothilone A (4) with a 0.5 M solution of

sodium hydroxide in MeOH–H₂O aﬀorded via a retro-aldol

reaction⁴⁶in 24% yield compound 34 and additionally, by

cleavage of the lactone bond, the acid 35 in 65% yield

(Scheme 6).

pound 37 was found. Introduction of TBS-groups [37

(71%) and 38

39

41⁴⁷(85%)] and subsequent selective CSA-

deprotection provided the primary alcohols [39

and 41

40 (79%)

42⁴³(88%), respectively].

Reaction of epoxide 35 with H₂SO₄in THF–H₂O, followed

by methyl ester formation with TMS–diazomethane gave 43 in

54% yield. Subsequent reaction with NaIO₄and then BH₄^Ϫion

exchange resin provided diol 38 (49%) and ester 44 (41%) which

Treatment of epoxide 34 with H₂SO₄in THF–H₂O provided

diol 36 which gave upon periodate oxidation and subsequent

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Scheme 6 Reagents: a) NaOH, MeOH; b) H₂SO₄; c) 1. NaIO₄, 2. BH₄^Ϫion exchange resin; d) TBSOTf, Et₃N; e) CSA; f ) 1. H₂SO₄, 2. TMS–CH₂N₂;

g) 1. NaIO₄2. BH₄^Ϫion exchange resin.

were separated by preparative HPLC. Alternatively, the diol 43

was cleaved to the carboxylic acids 45 and 46 in 86% and 49%

yields, respectively, by reaction with sodium periodate and sub-

sequent oxidation of the aldehyde fragments with sodium

chlorite in the presence of 2-methylbut-2-ene as a HOCl scav-

enger⁴⁸(Scheme 7). Whereas compounds 44 and 46 were

Scheme 8 Reagents: a) 1. CH₂N₂, 2. TBSOTf, Et₃N, 3. O₃, 4. NaBH₄.

Scheme 9 Reagents: a) 1. TBSCl, imidazole, 2. O₃, 3. Me₂S, 4. NaBH₄.

Scheme 7 Reagents: a) 1. NaIO₄, NaClO₂, 2-methylbut-2-ene.

readily traceable by analytical HPLC using a light scattering

detector, fragment 37 was hardly detected and analytical TLC

was required.

imidazole followed by cleavage of the ∆^17,18double bond by

ozonolysis (O₃–Me₂S) and subsequent reduction with sodium

borohydride aﬀorded lactone 48 in 80% yield.

The antifungal compound ambruticin S (5)^29,30(Scheme 8)

was esteriﬁed with diazomethane and the C-5 and C-6 hydroxy

groups were protected as TBS-ethers by treatment with tert-

butyldimethylsilyl triﬂuoromethanesulfonate (TBSOTf ) in the

presence of triethylamine. Subsequent ozonolysis followed by

sodium borohydride reduction aﬀorded the primary alcohol

47 in 40% overall yield.

Thus, selective fragmentation of a series of natural products

from myxobacteria and subsequent chemical modiﬁcations

gave access to twenty-three diverse primary alcohol building

blocks. Fragmentation reactions used so far cover the cleavage

of double bonds either by ozonolysis or OsO₄-oxidation and

subsequent oxidative cleavage with NaIO₄, lactone ring opening

by either hydrolysis or reductive cleavage, and acid-catalysed

epoxide hydrolysis followed by oxidative diol cleavage. The con-

version to carboxylic acids was achieved by either oxidation of

primary alcohol building blocks or by aldehyde oxidation. The

The cytostatic macrolide apicularen A (6)^31,32provided the

10-membered lactone fragment 48 by ozonolysis of the TBS-

protected derivative (Scheme 9). Reaction of 6 with TBSCl–

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Scheme 10 SPOT synthesis of a natural product-derived hybrid structure on a photolinker-modiﬁed APEG membrane. Reagents: (a) amino acid

coupling (5 equiv.): HOBt, DIC, NMP, (b) coupling of natural product-derived fragments (7–8 equiv.): p-nitrophenyl chloroformate, DMAP,

pyridine, NMP (yield: >80%).

application of other fragmentation strategies and the intro-

duction of diﬀerent functional groups for solid-phase coupling

reactions are presently under investigation.

performed by three reaction cycles of Fmoc-deprotection with

20% piperidine in DMF and subsequent amino acid coupling.

Rapid and nearly quantitative transformations were achieved

by using reactants in large excess. The precise spot loading was

determined to be 170 nmol by base-catalysed cleavage of the

Fmoc-group of the Phe–Phe–Amp tripeptide and subsequent

quantiﬁcation of the dibenzofulvene–piperidine adduct by

UV.⁵⁰The phenylalanine residues were introduced to simplify

the analysis of intermediates by RP-HPLC and MALDI-MS

providing a UV-label and additional mass. In library synthesis

they will be omitted or replaced by other amino acids to

increase diversity. The orthogonal Boc and Fmoc protecting

groups for the amino-functions at the 4-aminoproline allow the

subsequent attachment of diﬀerent natural product-derived

fragments. First, the Boc protecting group of the 4-amino-

proline was removed with 50% TFA. The sorangicin-derived

fragment 33 was reacted with p-nitrophenyl chloroformate and

DMAP in a 1 : 1 (v/v) mixture of pyridine and NMP and was

subsequently coupled to give a carbamate linkage. After

Fmoc-cleavage, the epothilone-derived building block 42 was

coupled to the second amino-function using the same coupling

procedure. The coupling reactions require a 7- to 8-fold excess

of the natural product fragment in order to reach a yield of

>80%. Then, all TBS-protecting groups were cleaved by reac-

tion with HF–pyridine and the product ﬁnally removed from

The solid-phase synthesis of natural product-derived hybrid

structures is exempliﬁed by selective chemical coupling of

sorangicin and epothilone fragments 33 and 42 via a single

SPOT-synthesis on a 100 nmol scale (Scheme 10). The combin-

ation of the natural product building blocks is achieved employ-

ing a membrane-linked spacer. To obtain a broad range of

molecular structures both the exchange of the natural prod-

uct building blocks and variation of the connective spacer is

intended in combinatorial library synthesis. Here, cis-4-amino-

-proline (Amp) is used as the spacer, which is attached via

two -phenylalanine (Phe) residues to a photolinker-derivatised

membrane. First, the Fmoc-protected aminoethyl photo-

linker⁴⁹was anchored to the commercially available amino-

functionalised polypropylene membrane by activation with

hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbo-

diimide (DIC) in N-methylpyrrolidin-2-one (NMP) followed

by capping of unreacted amino functions by acetylation with

acetic anhydride. The initial spot loading is determined by the

given membrane loading (600 nmol cm^Ϫ2), the spot size (1.2 µl

per spot, ∼0.28 cm²), and the fact that the photolinker was

added in a threefold excess. Stepwise elongation by two

-phenylalanine residues and the 4-amino--proline spacer was

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J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

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the solid-support by exposure to UV light at 365 nm with an

average product recovery of 80%. Analysis by reverse-phase

HPLC ESI-MS revealed that compound 49 with a molecular

mass of 876 was formed as the main product (70%) as deter-

mined by peak area integration of the HPLC chromatograms at

254 nm and 211 nm, respectively. Besides compound 49 a by-

product (10%) and a number of very minor components, which

add up to about 20% were detected. According to the molecular

mass of 901 the by-product contains two epothilone fragments

linked to the aminoproline spacer and is thus formed due to an

incomplete reaction of the sorangicin fragment 33 in the ﬁrst

coupling step. Based on an initial spot loading of 170 nmol

and a product recovery of 80% from the solid-support we

can calculate that ∼100 nmol of compound 49 were formed

corresponding to an overall yield of 60%.

In summary, we have prepared a collection of natural

product-derived chiral building blocks for the synthesis of

combinatorial compound libraries with high structural diver-

sity. The applicability of the primary alcohol fragments in

solid-phase synthesis was exemplarily demonstrated with the

SPOT-synthesis technique. The building blocks are currently

being used for the automated highly parallel 10 nmol-scale

synthesis of natural product hybrid structures, which will be

tested for biological activity using a miniaturised ultra-high

throughput screening system.

obtained from Neosystem (Strasbourg, France). Photolytic

cleavage was performed using a Vilber Lourmat TFP-35.L

transilluminator at 365 nm.

(2R,3S,8S )-8-(tert-Butyldimethylsilyloxy)-2,3-dimethoxy-8-

phenyloctan-1-ol 8 and (2R,3R,4S,5S,6S )-2-[(1R)-2-(tert-

butyldimethylsilyloxy)-1-methylethyl]-4-(tert-butyldimethyl-

silyloxy)-6-[(1S )-2-hydroxy-1-methylethyl]-3-methoxy-5-

methyltetrahydro-2H-pyran-2-ol 9 and

Soraphen A (1) (500 mg, 0.97 mmol) was added at 0 ЊC to

a solution of LiAlH₄(220 mg, 5.80 mmol) in anhydrous THF

(10 ml). The reaction mixture was stirred under a N₂atmos-

phere for 6 h at room temperature, then treated at 0 ЊC with 1 M

HCl and extracted three times with ethyl acetate. The combined

organic layers were washed with saturated aqueous sodium

hydrogen carbonate, then with brine, dried over MgSO₄, and

concentrated in vacuo. The crude product was dissolved

in anhydrous DMF (5 ml) containing tert-butyldimethylsilyl

chloride (1.38 g, 9.16 mmol) and imidazole (0.63 g, 9.25 mmol).

After stirring for 48 h at room temperature, the reaction mix-

ture was diluted with water and extracted three times with

diethyl ether. The combined organic layers were washed with

H₂O, dried over Na₂SO₄, and concentrated in vacuo. Puriﬁ-

cation of the residue by column chromatography (petroleum

ether–ethyl acetate 14 : 1) gave 1,5,17-tris(tert-butyldimethyl-

silyloxy)-1,17-secosoraphen 7³⁶(492 mg, 59%) as a colourless

oil; R_f0.65 (petroleum ether–ethyl acetate 5 : 1). Compound 7

(241 mg, 0.277 mmol) was dissolved in CH₂Cl₂(16 ml) and

ozone was introduced at Ϫ70 ЊC for 5 min. After addition of

MeOH (16 ml), the reaction mixture was treated at 0 ЊC with

NaBH₄(35 mg, 0.93 mmol) and stirred for 1.5 h at room

temperature. The reaction mixture was then treated with a

1 M aqueous solution of NaH₂PO₄–K₂HPO₄(pH 7) and

extracted three times with CH₂Cl₂. The combined organic

layers were washed with H₂O, dried over MgSO₄, and concen-

trated in vacuo. Puriﬁcation of the residue by column chrom-

atography (petroleum ether–ethyl acetate 5 : 1, then 2 : 1) gave 8

(73.0 mg, 66%) and 9 (78.2 mg, 57%) as colourless oils.

Experimental

General methods

Analytical TLC was performed on silica gel Si 60 F₂₅₄alu-

minium sheets, 0.2 mm (Merck). Products were visualised by

either UV absorption at 254 nm or staining with cerium()

sulfate–phosphomolybdic acid in sulfuric acid followed by

charring. Silica gel 60 (0.063–0.200 mm) was used for prepar-

ative column chromatography and precoated silica gel Si 60

F₂₅₄plates of 0.25, 0.5, 1.0 or 2.0 mm layer thickness

for preparative TLC. Analytical HPLC was carried out on

250 × 4 Nucleosil 100–7 C-18 (Column A) and Nucleosil

100–7 (Column B) columns (Machery-Nagel) with detection by

either UV absorption or light scattering. Preparative HPLC

was carried out on 16 × 250 columns of either Nucleosil

100–7 C-18, Machery-Nagel (Column A) or Nucleosil 100–7,

Knauer (Column B). Optical rotations were measured on a

Perkin-Elmer 241 polarimeter. [α]_Dvalues are given in units of

10^Ϫ1deg cm²g^Ϫ1. IR spectra were recorded from samples in

KBr pellets on a Nicolet 20DXB FT-IR spectrometer. NMR

spectra were measured on Bruker AM-300 [300 MHz (¹H); 75.5

MHz (¹³C)] or Bruker WM-400 [(400 MHz (¹H); 100.6 MHz

(¹³C)]. J values are given in Hz. High-resolution mass spectra

(HRMS) were measured on a MAT 95 Finnigran (DCI)

or Kratos MS 50 RFTC (FAB) spectrometer from peak

matching M/∆M = 10000. Elemental analyses were carried

out by Mikroanalytisches Laboratorium I. Beetz (Kronach,

Germany). HPLC-MS was performed on ET 125/2 Nucleosil

120–5 C-18, Machery-Nagel, Hewlett-Packard 1050 diode

array detector, PE Sciex Api-2000 LC/MS/MS. THF was

distilled from sodium–benzophenone and CH₂Cl₂from

8. R_f(petroleum ether–ethyl acetate 5 : 1) 0.11; [α]²_D²Ϫ24.5

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13465m, 2930s, 2857s, 1472m,

1257m, 1111s, 1066m, 837s, 775m, 700m; δ_H(400 MHz; CDCl₃)

Ϫ0.16, 0.01 [6 H, 2 × s, Si(CH₃)₂], 0.87 [9 H, s, C(CH₃)₃], 3.15

(1 H, dt, J 4.6 and 4.6, 2-H), 3.29 (1 H, m, 3-H), 3.39, 3.41 (6 H,

2 × s, 2-OMe, 3-OMe), 3.70 (2 H, m, 1-H₂), 4.63 (1 H, dd, J 5.0

and 7.1, 8-H), 7.18–7.31 (5 H, m, C8-Phenyl); δ_C(100.6 MHz;

CDCl₃) Ϫ4.9, Ϫ4.6 [Si(CH₃)₂], 18.2 [C(CH₃)₃], 25.9 [C(CH₃)₃],

25.4, 25.7, 30.8, 40.9 (C-4, C-5, C-6, C-7), 57.8, 58.6 (2-Me,

3-OMe), 61.0 (C-1), 75.0 (C-8), 81.6 (C-3), 82.6 (C-2), 125.9,

126.8, 128.0, 145.8 (8-Phenyl); m/z (FAB) 419 (M ϩ Na^ϩ,

100%), 265 [(M – TBSO) ϩ Na^ϩ, 65]; HRMS found (M ϩ Na^ϩ)

419.2594; C₂₂H₄₀O₄Si requires 419.2582.

9. R_f0.47 (petroleum ether–ethyl acetate 5 : 1); [α]²_D²Ϫ3.5

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13449m, 2954s, 2930s, 2886m,

2857m, 1472m, 1255m, 1111m, 1030s, 838s, 778m; δ_H(300

MHz; CDCl₃) 0.03, 0.04, 0.11, 0.13 [12 H, 4 × s, 2 × Si(CH₃)₂],

0.79 (3 H, d, J 6.8, CHCH₃), 0.87, 0.90 [18 H, 2 × s,

2 × C(CH₃)₃], 0.97 (3 H, d, J 6.8, CHCH₃), 1.03 (3 H, d, J 7.1,

5-Me), 1.70 (1 H, m, 5-H), 1.93 (1 H, m, CHCH₃), 2.10 (1 H, m,

CHCH₃), 2.97 (1 H, dd, J 1.1 and 3.0, 3-H), 3.37 (3 H, s,

3-OMe), 3.51 (1 H, dd, J 9.4 and 9.7, CH_2a), 3.63 (2 H, m, CH₂),

3.91 (1 H, dd, J 3.4 and 9.7, CH_2b), 3.99 (1 H, dd, J 2.6 and

10.5, 6-H), 4.08 (1 H, dd, J 2.6 and 2.6, 4-H); δ_C(100.6 MHz;

CDCl₃) Ϫ5.4, Ϫ5.3, Ϫ5.1, Ϫ5.0 [2 × Si(CH₃)₂], 10.8 (5-Me),

11.8, 13.1 (2 × CHCH₃), 18.3, 18.0 [2 × C(CH₃)₃], 25.7, 26.0

[2 × C(CH₃)₃], 35.4 (C-5), 36.1, 41.3 (2 × CHCH₃), 57.5

(3-OMe), 63.5, 67.7 (2 × CH₂), 70.5, 70.7 (C-4, C-6), 77.9 (C-3),

100.5 (C-2); m/z (FAB) 529 (M ϩ Na^ϩ, 40%), 489 [(M – H₂O) ϩ

H^ϩ, 100]; HRMS found (M ϩ Na^ϩ) 529.3357; C₂₅H₅₄O₆Si₂

requires 529.3349.

CaH₂prior to use. Ozonolysis was performed with

a

Sander Labor-Ozonisator. Polymer-supported borohydride on

Ϫ

Amberlite^®IRA-400 (∼2.5 mmol BH₄g^Ϫ1resin) and HF–

pyridine complex were supplied by Sigma-Aldrich Chemie

GmbH (Taufkirchen, Germany). The primary alkyl amino-

derivatised polypropylene APEG-membrane (loading 600 nmol

cm^Ϫ2) was obtained from AIMS Scientiﬁc Products GmbH

(Braunschweig, Germany). N ^α-Fmoc--phenylalanine (Fmoc–

Phe–OH) and the 4-{4-[1-(Fmoc-amino)ethyl]-2-methoxy-5-

nitrophenoxy}butanoic acid photolinker were purchased from

Calbiochem-Novabiochem GmbH (Bad Soden, Germany).

Protected cis-4-amino--proline [Boc--Amp(Fmoc)-OH] was

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

2495

View Article Online

Triethylammonium (2R,3S,8S )-8-(tert-butyldimethylsilyloxy)-

2,3-dimethoxy-8-phenyloctanoate 10

2.06 (1 H, m, 2-H), 2.43 (1 H, m, 8-H), 3.08–3.15 (2 H, m, 4-H,

12-H), 3.25, 3.37, 3.41 (12 H, 3 × s, 4-OMe, 5-OMe, 11-OMe,

12-OMe, 3.45 (1 H, m, 5-H), 3.54 (1 H, dd, J 3.6 and 8.6, 11-H),

3.69 (1 H, dd, J 2.6 and 9.7, 1-H_a), 3.93 (1 H, dd, J 1.5 and 8.7,

7-H), 4.07 (1 H, dd, J 5.0 and 9.7, 1-H_b), 4.60 (1 H, dd, J 5.1 and

7.6, 17-H), 7.15–7.30 (5 H, m, C17-Phenyl); δ_C(100.6 MHz;

CDCl₃) Ϫ5.3, Ϫ5.2, Ϫ4.9, Ϫ4.6 [2 × Si(CH₃)₂], 11.1, 11.9

(2-Me, 8-Me), 16.7 (6-Me), 18.2, 18.3 [2 × C(CH₃)₃], 25.7, 25.8

(C-14, C-15), 25.9, 26.0 [2 × C(CH₃)₃], 30.6 (C-13), 31.1 (C-6),

37.3 (C-8), 39.4 (C-2), 41.0 (C-16), 56.4, 57.4, 57.7, 58.5

(4-OMe, 5-OMe, 11-OMe, 12-OMe), 64.8 (C-1), 70.0 (C-7),

75.1 (C-17), 76.4 (C-4), 79.6 (C-5), 84.0 (C-12), 84.6 (C-11),

100.3 (C-3), 125.9, 126.7, 128.0, 146.0 (C-10, 17-Phenyl), 140.0

(C-9); m/z (FAB) 789 (M ϩ Na^ϩ, 15%), 157 (70), 73 (100);

HRMS found (M ϩ Na^ϩ) 789.5176; C₄₂H₇₈O₈Si₂requires

789.5133.

Compound 8 (5.0 mg, 12.6 µmol) was added to a solution of

pyridinium dichromate (PDC) (42 mg, 0.112 mmol) in

anhydrous DMF (250 µl). The reaction mixture was stirred for

40 h at room temperature, then diluted with ethyl acetate,

washed with brine, dried over MgSO₄, and concentrated

in vacuo. Puriﬁcation of the residue by column chromatography

(CH₂Cl₂–MeOH 90 : 10, 1% triethylamine) gave 10 (3.9 mg,

61%); R_f0.34 (CH₂Cl₂–MeOH 90 : 10); δ_H(300 MHz; CD₃OD)

Ϫ0.12, 0.08 [6 H, 2 × s, Si(CH₃)₂], 0.92 [9 H, s, C(CH₃)₃], 1.35

[9 H, t, N(CH₂CH₃)₃], 3.24 [6 H, q, N(CH₂CH₃)₃], 3.41, 3.42

(6 H, 2 × s, 2-OMe, 3-OMe), 3.48 (1 H, m, 3-H), 3.95 (1 H, d,

J 3.4, 2-H), 4.72 (1 H, dd, J 5.3 and 7.6, 8-H), 7.20–7.34 (5 H,

m, C8-Phenyl); δ_C(100.6 MHz; CDCl₃) Ϫ4.7, Ϫ4.4 [Si(CH₃)₂],

9.2 [N(CH₂CH₃)₃], 19.0 [C(CH₃)₃], 26.3 [C(CH₃)₃], 47.9

[N(CH₂CH₃)₃], 26.6, 26.7, 31.2, 41.9 (C-4, C-5, C-6, C-7), 58.1,

58.9 (2-OMe, 3-OMe), 76.4 (C-8), 83.0 (C-2), 83.6 (C-3), 127.0,

128.0, 129.1, 147.0 (8-Phenyl), 175.1 (C-1); m/z (FAB) 409

(M Ϫ H^Ϫ, 15%), 305 (40), 153 (100); HRMS found (M Ϫ H^Ϫ)

409.2373; C₂₂H₃₈O₅Si requires 409.2410.

(2R,3S,8S )-8-(tert-Butyldimethylsilyloxy)-2,3-dimethoxy-8-

phenyloctan-1-ol 8 and (2R,3R,4S,5S,6S )-2-[(1R)-2-(tert-

butyldimethylsilyloxy)-1-methylethyl]-3,4-dimethoxy-6-[(1S )-2-

hydroxy-1-methylethyl]-5-methyltetrahydro-2H-pyran-2-ol 12

Compound 11 (200 mg, 0.262 mmol) was dissolved in CH₂Cl₂

(15 ml) and ozone was introduced at Ϫ75 ЊC for 5 min. After

addition of MeOH (13 ml), the reaction mixture was treated at

0 ЊC with NaBH₄(33 mg, 0.87 mmol) and stirred for 1.5 h at

room temperature. The reaction mixture was then treated with

a 1 M aqueous solution of NaH₂PO₄–K₂HPO₄(pH 7) and

extracted three times with ethyl acetate. The combined organic

layers were washed with H₂O, dried over MgSO₄, and concen-

trated in vacuo. Puriﬁcation of the residue by column chrom-

atography (hexane–ethyl acetate 3 : 1) gave 8 (75.5 mg, 73%)

and 12 (19.4 mg, 18%) as colourless oils.

1,17-Bis(tert-butyldimethylsilyloxy)-5-methoxy-1,17-seco-

soraphen 11

Soraphen A (1) (395 mg, 0.89 mmol) was dissolved in

anhydrous hexamethylphosphoramide (HMPA) (5 ml) under

a N₂atmosphere and subsequently treated with iodomethane

(1.0 ml, 16 mmol) and sodium hydride (80%, 200 mg, 6.7

mmol). The reaction mixture was stirred for 25 min at 55–60 ЊC,

then cooled to 5 ЊC, treated with a 1 M aqueous solution of

NaH₂PO₄–K₂HPO₄(pH 7), and extracted with diethyl ether.

The organic phase was washed with NaH₂PO₄–K₂HPO₄(pH 7),

dried over MgSO₄, and concentrated in vacuo. Puriﬁcation of

the residue by column chromatography (petroleum ether–ethyl

acetate 3 : 1) gave 3,5-di-O-methylsoraphen (403 mg, 83%) as a

colourless oil; R_f0.37 (petroleum ether–ethyl acetate 2 : 1). 3,5-

Di-O-methylsoraphen (185 mg, 0.33 mmol) was dissolved in

THF (12.2 ml) and 1 M HCl (6.1 ml), and stirred for 3 days at

room temperature. THF was removed in vacuo, the reaction

mixture was treated with a 5% aqueous solution of sodium

hydrogen carbonate, and was extracted three times with ethyl

acetate. The combined organic layers were washed with a 1 M

aqueous solution of NaH₂PO₄–K₂HPO₄(pH 7), dried over

MgSO₄, and concentrated in vacuo. Puriﬁcation of the residue

by column chromatography (petroleum ether–ethyl acetate

3 : 1) gave 5-O-methylsoraphen (180 mg, 98%) as a colourless

oil; R_f0.33 (petroleum ether–ethyl acetate 2 : 1).

A solution of 5-O-methyl-soraphen (245 mg, 0.46 mmol)

in anhydrous THF (2 ml) was added to a solution of LiAlH₄

(110 mg, 2.90 mmol) in anhydrous THF (2 ml). The reaction

mixture was stirred under a N₂atmosphere for 4.5 h at room

temperature, was then treated at 0 ЊC with 1 M HCl, and was

extracted three times with ethyl acetate. The combined organic

layers were washed with saturated aqueous sodium hydrogen

carbonate, then with brine, dried over MgSO₄, and concen-

trated in vacuo. The crude product was dissolved under N₂

atmosphere in anhydrous DMF (2.8 ml) containing tert-

butyldimethylsilyl chloride (685 mg, 4.54 mmol) and imidazole

(312 mg, 4.58 mmol). After stirring for 72 h at room temper-

ature, the reaction mixture was diluted with water and extracted

three times with diethyl ether. The combined organic layers

were washed with H₂O, dried over MgSO₄, and concentrated

in vacuo. Puriﬁcation of the residue by column chromatography

(petroleum ether–ethyl acetate 10 : 1) gave 11 (201 mg, 58%) as

a colourless oil; R_f0.46 (petroleum ether–ethyl acetate 6 : 1);

ν_max(KBr)/cm^Ϫ13479w, 2930s, 2857s, 1472m, 1257m, 1090s,

837s, 775m; δ_H(400 MHz; CDCl₃) 0.86, 0.87 [18 H, 2 × s,

2 × C(CH₃)₃], 0.95 (3 H, d, J 6.6, 8-Me), 1.00, 1.01 (6 H, 2 × d,

2-Me, 6-Me), 1.54–1.75 (2 H, m, 16-H₂), 1.92 (1 H, m, 6-H),

12. R_f0.19 (hexane–ethyl acetate 3 : 1); [α]²_D²Ϫ24.9 (c 1.0 in

CHCl₃); δ_H(400 MHz; CDCl₃) 0.06, 0.07 [6 H, 2 × s, Si(CH₃)₂],

0.83 (3 H, d, J 6.8, CHCH₃), 0.88 [9 H, s, C(CH₃)₃], 1.01 (3 H, d,

J 7.4, CHCH₃), 1.04 (3 H, d, J 7.1, 5-Me), 2.10 (1 H, m,

CHCH₃), 3.13 (1 H, dd, J 1.1 and 2.3, 3-H), 3.38, 3.42 (6 H,

2 × s, 3-OMe, 4-OMe); m/z (FAB) 429 (M ϩ Na^ϩ, 100%), 389

[(M Ϫ H₂O) ϩ H^ϩ, 40]; HRMS found (M ϩ Na^ϩ) 429.2695;

C₂₀H₄₂O₆Si requires 429.2648.

Preparation of ꢀ-lactone 13

Soraphen A (1) (1.0 g, 1.92 mmol) was dissolved in MeOH

(44 ml) and treated with 95% H₂SO₄(1.3 ml). After stirring for

24 h at room temperature, the reaction mixture was concen-

trated in vacuo, then treated with a 5% aqueous solution of

sodium hydrogen carbonate and extracted three times with

ethyl acetate. The combined organic layers were washed with a

1 M aqueous solution of NaH₂PO₄–K₂HPO₄(pH 7), then with

brine, dried over MgSO₄, and concentrated in vacuo. Puriﬁ-

cation of the residue by column chromatography (petroleum

ether–ethyl acetate 5 : 2, then 1 : 1) gave 13³⁵(858 mg, 81%) as a

colourless oil; R_f0.72 (CH₂Cl₂–acetone 90 : 10); [α]²_D⁴Ϫ69.4

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ12980s, 2945s, 2840m, 2820m,

1795s, 1465s, 1460s, 1380m, 1360m, 1330m, 1270m, 1215m,

1195m, 1100m, 1030m, 1005m, 975m, 920m, 760m, 720m, 705s;

δ_H(300 MHz; CDCl₃) 0.95 (6 H, d, J 7.0, 6-Me, 8-Me), 1.31

(3 H, J 7.2, 2-Me), 1.20–1.45 (6 H, m, 13-H₂, 14-H₂, 15-H₂),

1.62 (1 H, m, 6-H_a), 1.77 (1 H, m, 6-H_b), 1.95–2.06 (2 H, m,

5-H_a, 6-H), 2.27 (1 H, m, 5-H_b), 2.38 (1 H, m, 8-H), 3.02 (1 H, q,

J 7.2, 2-H), 3.12 (1 H, m, 12-H), 3.18 (17-OMe), 3.23 (11-OMe),

3.36 (12-OMe), 3.39 (4-OMe), 3.40 (3-OMe), 3.49 (1 H, m,

11-H), 5.43 (1 H, dd, J 8.2 and 15.6, 10-H), 5.68 (1 H, dd, J 7.7

and 15.6, 9-H), 7.24–7.35 (5 H, m, 8-Phenyl); δ_C(75.5 MHz;

CDCl₃) 9.2 (2-Me), 12.3 (8-Me), 16.4 (6-Me), 25.8, 26.0 (C-14,

C-15), 27.9 (C-6), 30.8 (C-13), 32.3 (C-5), 38.2 (C-8, C-16),

42.2 (C-2), 50.0 (4-OMe), 51.4 (3-OMe), 56.5 (11-OMe), 56.6

(17-OMe), 58.7 (12-OMe), 74.8 (C-7), 83.7 (C-12), 84.1 (C-17),

84.8 (C-11), 101.2 (C-3), 102.2 (C-4), 127.4 (C-10), 138.6 (C-9),

2496

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

View Article Online

126.7, 127.5, 128.4, 142.5 (8-Phenyl), 175.8 (C-1); m/z (DCI)

566 (M ϩ NH₄^ϩ, 100%), HRMS found (M ϩ NH₄^ϩ) 566.3620;

C₁₄H₂₄O₆requires 566.3693 (Found: C, 67.80; H, 8.8. Calc. for

C₃₁H₄₈O₈: C, 67.84; H 8.8%).

2-methylbut-2-ene (900 µl) and a solution of sodium chlorite

(81 mg) and NaH₂PO₄(61 mg) in H₂O (1.2 ml) were added.

After stirring for 1.5 h at 0 ЊC, the reaction was quenched by the

addition of a 10% aqueous solution of Na₂SO₃. Saturated

aqueous NH₄Cl was added and the resulting mixture was

extracted three times with ethyl acetate. The combined organic

layers were washed with brine, dried over MgSO₄, and concen-

trated in vacuo. Puriﬁcation of the residue by column chrom-

atography (CH₂Cl₂–MeOH 95 : 5, 1% triethylamine) gave 16

(37.2 mg, 60%); R_f0.24 (CH₂Cl₂–MeOH 90 : 10); ν_max(KBr)/

cm^Ϫ12936s, 2860m, 2825m, 2677m, 1747s, 1454m, 1190m,

1113s, 761m, 702s; δ_H(300 MHz; CD₃OD) 1.35 (9 H, t,

NCH₂CH₃), 1.81 (1 H, m, 7-H_a), 3.21 (3 H, s, 8-OMe), 3.25

(6 H, q, NCH₂CH₃), 3.42, 3.41 (6 H, 2 × s, 2-OMe, 3-OMe),

3.47 (1 H, m, 3-H), 3.94 (1 H, d, J 3.8, 2-H), 4.16 (1 H, t, J 7.1,

8-H); δ_C(100.6 MHz; CD₃OD) 9.2 (NCH₂CH₃), 26.6, 26.7

(C-5, C-6), 31.1 (C-4), 38.9 (C-7), 47.9 (NCH₂CH₃), 56.8

(8-OMe), 58.1, 58.9 (2-OMe, 3-OMe), 82.9 (C-2), 83.5 (C-3),

85.3 (C-8), 127.8, 128.7, 129.4 (8-Phenyl), 175.0 (C-1); m/z

(DCI) 328 (M ϩ NH₄^ϩ, 100%), 127 (60), 110 (100); HRMS

found (M ϩ NH₄^ϩ) 328.2131; C₁₇H₂₆O₅requires 328.2124.

(2R,3S,8R)-2,3,8-Trimethoxy-8-phenyloctan-1-ol 14 and

bicyclic lactone 15

Compound 13 (86.4 mg, 0.157 mmol) was dissolved in acetone

(1.6 ml) and H₂O (170 µl). 4-Methylmorpholine N-oxide

(NMO) (85 mg, 4.0 equiv.) was added, followed by OsO₄

(300 µl, 2.5 wt% solution in tert-butanol, 0.15 equiv.). The reac-

tion mixture was stirred for 24 h at room temperature, then the

solvent was removed with a ﬂow of nitrogen, the residue was

partitioned between ethyl acetate and H₂O, and the aqueous

layer extracted twice with ethyl acetate. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. The

residue was dissolved in THF (1.7 ml) and treated with a solu-

tion of NaIO₄(130 mg, 0.60 mmol) in H₂O (800 µl). After

stirring for 40 min at room temperature, the organic solvent was

removed in vacuo, brine was added, and the reaction mixture

was extracted three times with ethyl acetate. The combined

organic layers were dried over MgSO₄and concentrated in

vacuo. The residue was dissolved in CH₂Cl₂(3 ml) and MeOH

(6 ml) and treated at 0 ЊC with NaBH₄(16 mg, 0.42 mmol).

After stirring for 2 h at room temperature, the reaction mixture

was treated with acetic acid and concentrated in vacuo. The

residue was partitioned between ethyl acetate and a 1 M aque-

ous solution of NaH₂PO₄–K₂HPO₄(pH 7), and the aqueous

layer extracted twice with ethyl acetate. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. Puri-

ﬁcation of the residue by column chromatography (petroleum

ether–ethyl acetate 1 : 1, then 1 : 2) gave 14 (33.7 mg, 72%) and

15 (33.6 mg, 74%) as colourless oils.

Oxidation of 15 to carboxylic acid 17

Compound 15 (24.1 mg, 0.084 mmol) was dissolved in

anhydrous DMF (200 µl) and treated with a solution of PDC

(285 mg, 0.76 mmol) in anhydrous DMF (1.5 ml). After stirring

for 27 h at room temperature, Florisil (activated magnesium

silicate) was added, the reaction mixture was diluted with ethyl

acetate, ﬁltered over silica gel, and concentrated in vacuo. Puri-

ﬁcation of the residue by column chromatography (CH₂Cl₂–

MeOH 95 : 5, 2% triethylamine) gave 17 (19.1 mg, 56%); R_f0.39

(CH₂Cl₂–MeOH 90 : 10); ν_max(KBr)/cm^Ϫ13429s, 2976s, 2940s,

2677s, 1791s, 1730m, 1463m, 1196m, 1142m, 1120s, 1027m,

973m, 954m; δ_H(300 MHz; CD₃OD) 0.96 (3 H, d, J 7.5,

6-Me), 1.11 (3 H, d, J 6.8, 8-Me), 1.32–1.38 (12 H, m, 2-Me,

NCH₂CH₃), 2.05–2.15 (2 H, m, 5-H_a, 6-H), 2.36 (1 H, m, 5-H_b),

2.53 (1 H, m, 8-H), 3.06 (1 H, q, J 7.1, 2-H), 3.24 (6 H, q,

NCH₂CH₃), 3.39, 3.46 (6 H, 2 × s, 3-OMe, 4-OMe), 4.06

(1 H, dd, J 1.9 and 10.6, 7-H); δ_C(100.6 MHz; CD₃OD)

9.25 (NCH₂CH₃), 9.4, 12.6, 13.9 (2-Me, 6-Me, 8-Me), 28.2

(C-6), 33.0 (C-5), 43.2 (C-2), 44.1 (C-8), 47.9 (NCH₂CH₃), 50.2,

52.2 (3-OMe, 4-OMe), 74.5 (C-7), 102.4, 103.7 (C-3, C-4),

177.7, 180.8 (C-1, C-9); m/z (DCI) 320 (M ϩ NH₄^ϩ, 100%);

HRMS found (M ϩ NH₄^ϩ) 320.1757; C₁₄H₂₂O₇requires

320.1710.

14. R_f0.28 (petroleum ether–ethyl acetate 1 : 1); [α]²_D²ϩ35.1

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13449m, 2936s, 2859m, 2823m,

1453m, 1108, 703m; δ_H(400 MHz; CDCl₃) 1.20–1.50 (6 H, m,

4-H₂, 5-H₂, 6-H₂), 1.64 (1 H, m, 7-H_a), 1.81 (1 H, m, 7-H_b), 3.14

(1 H, dt, J 4.6 and 4.6, 2-H), 3.28 (1 H, m, 3-H), 3.19 (3 H, s,

8-OMe), 3.38 (3 H, s, 3-OMe), 3.41 (3 H, s, 2-OMe), 3.70 (2 H,

m, 1-H₂), 4.07 (1 H, t, J 6.1, 8-H), 7.24–7.35 (5 H, m, 8-Phenyl);

δ_C(100.6 MHz; CDCl₃) 25.4, 26.0, 30.8, 38.1 (C-4, C-5, C-6,

C-7), 56.7 (8-OMe), 57.8, 58.6 (2-OMe, 3-OMe), 61.0 (C-1),

81.6 (C-3), 82.6 (C-2), 84.1 (C-8), 125.9, 126.8, 128.0, 145.8

ϩ

ؒ

(8-Phenyl); m/z (EI) 296 ([M ] , 40%), 237 (100); HRMS found

ϩ

ؒ

[M ] 296.1995; C₁₇H₂₈O₄requires 296.1988 (Found: C, 68.82;

H, 9.5. Calc. for C₁₇H₂₈O₄: C, 68.87; H 9.5%).

15. R_f0.16 (petroleum ether–ethyl acetate 1 : 1); [α]²_D²Ϫ111.9

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13533m, 2966m, 2946m, 1793s,

1460m, 1271m, 1197m, 1117s, 1028s, 971s, 951m; δ_H(400 MHz;

CDCl₃) 0.87 (3 H, d, J 7.1, 8-Me), 0.95 (3 H, d, J 7.1, 6-Me),

1.35 (3 H, d, J 7.1, 2-Me), 1.86 (1 H, m, 8-H), 1.98–2.05 (2 H,

m, 5-H_a, 6-H), 2.28 (1 H, m, 5-H_b), 3.07 (1 H, q, J 7.1, 2-H),

3.39 (3 H, s, 4-OMe), 3.48 (3 H, s, 3-OMe), 3.65–3.77 (3 H, m,

7-H, 9-H₂); δ_C(100.6 MHz; CDCl₃) 9.3 (2-Me), 12.3 (8-Me),

12.8 (6-Me), 27.8 (C-6), 32.0 (C-5), 36.5 (C-8), 41.9 (C-2), 50.0

(4-OMe), 51.5 (3-OMe), 66.6 (C-9), 74.6 (C-7), 101.5 (C-3),

102.2 (C-4), 175.5 (C-1); m/z (DCI) 306 (M ϩ NH₄^ϩ, 100%);

HRMS found (M ϩ NH₄^ϩ) 306.1967; C₁₄H₂₄O₆requires

306.1917 (Found: C, 58.36; H, 8.4. Calc. for C₁₄H₂₄O₆: C, 58.30;

H 8.4%).

(1RS )-1-[4-(Hydroxymethyl)-2,4Ј-bi-1,3-thiazol-2Ј-yl]ethanol

18 and methyl (2E,4R,5R)-6-hydroxy-3,5-dimethoxy-4-methyl-

hex-2-enoate 19

Myxothiazol Z (2b)⁴²(576 mg, 1.14 mmol) was dissolved

in acetone (11.3 ml) and H₂O (1.2 ml). 4-Methylmorpholine

N-oxide (NMO) (619 mg, 0.03 equiv.) was added, followed by

OsO₄(435 µl, 2.5 wt% solution in tert-butanol). The reaction

mixture was stirred for 24 h at room temperature, then the sol-

vent was removed with a ﬂow of nitrogen, the residue was par-

titioned between ethyl acetate and brine, and the aqueous layer

was extracted twice with ethyl acetate. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. The

residue was dissolved in THF (15.3 ml) and treated with a

solution of NaIO₄(1.10 g, 5.1 mmol) in H₂O (7.6 ml). After

stirring for 40 min at room temperature, the organic solvent

was removed in vacuo, brine was added, and the reaction mix-

ture was extracted three times with ethyl acetate. The combined

organic layers were dried over MgSO₄and concentrated in

vacuo. The residue was dissolved in CH₂Cl₂(20 ml) and MeOH

(40 ml), and treated at 0 ЊC with NaBH₄(290 mg, 7.67 mmol).

After stirring for 2 h at room temperature, the reaction

mixture was treated with acetic acid and concentrated in vacuo.

The residue was partitioned between ethyl acetate and a 1 M

Triethylammonium (2R,3S,8R)-2,3,8-trimethoxy-8-phenyl-

octanoate 16

Compound 14 (45.1 mg, 0.15 mmol) was dissolved in acetone

(470 µl) and treated at 0 ЊC with a 5% aqueous solution of

sodium hydrogen carbonate (470 µl), containing KBr (1.9 mg,

0.015 mmol). 2,2,6,6-Tetramethylpiperidine-1-oxyl (36 mg,

0.23 mmol) was added, followed by the dropwise addition of

sodium hypochlorite (aqueous 4–6%, 400 µl) over 20 min. Then

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

2497

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aqueous solution of NaH₂PO₄–K₂HPO₄(pH 7), and the

aqueous layer extracted twice with ethyl acetate. The com-

bined organic layers were dried over MgSO₄and concentrated

in vacuo. Puriﬁcation of the residue by column chromatography

(CH₂Cl₂–MeOH 95 : 5, 93 : 7, then 90 : 10) gave 18 (215 mg,

78%) and 19 (167 mg, 67%) as colourless oils.

(2 × SCH), 149.0, 157.2, 163.6, 179.1 (4 × C); m/z (DCI) 357

(M ϩ H^ϩ, 100%); HRMS found (M ϩ H^ϩ) 357.1103; C₁₅H₂₄-

N₂O₂S₂Si requires 357.1127.

2Ј-[(1RS )-1-(tert-Butyldimethylsilyloxy)ethyl]-2,4Јbi-1,3-

thiazole-4-carboxylic acid 22

18. R_f0.12 (CH₂Cl₂–MeOH 95 : 5); ν_max(KBr)/cm^Ϫ13309s,

3113s, 2982m, 2930m, 1539m, 1435m, 1362m, 1307m, 1176s,

1139s, 1027s, 1018s, 964m, 802m, 773m; δ_H(400 MHz; CD₃OD)

1.64 (3 H, d, J 6.6, CHCH₃), 4.77 (2 H, s, CH₂O), 5.12 (1 H,

q, J 6.6, CHCH₃), 7.44, 8.06 (2 H, 2 × s, 2 × SCH); δ_C(100.6

MHz; CD₃OD) 26.7 (CHCH₃), 63.6 (CH₂O), 71.2 (CHCH₃),

118.9, 119.5 (2 × SCH), 152.3, 161.5, 167.1, 182.8 (4 × C);

m/z (DCI) 260 (M ϩ NH₄^ϩ, 100%), 243 (M ϩ H^ϩ, 100);

HRMS found (M ϩ NH₄^ϩ) 260.0532; C₉H₁₀N₂O₂S₂requires

260.0527.

Compound 21 (23.8 mg, 0.067 mmol) was dissolved in acetone

(400 µl) and treated at 0 ЊC with a 5% aqueous solution of

sodium hydrogen carbonate (200 µl), containing KBr (0.8 mg).

2,2,6,6-Tetramethylpiperidine-1-oxyl (16 mg, 0.115 mmol) was

added, followed by the dropwise addition of sodium hypo-

chlorite (aqueous 4–6%, 300 µl) over 15 min. After stirring for

40 min at room temperature, a second portion of sodium

hypochlorite (aqueous 4–6%, 300 µl) was added dropwise over

15 min at 0 ЊC and the reaction mixture was stirred for 2 h at

room temperature. Saturated aqueous NH₄Cl was added and

the resulting mixture was extracted three times with ethyl acet-

ate. The combined organic layers were washed with brine, dried

over MgSO₄, and concentrated in vacuo. Puriﬁcation of the

19. R_f0.35 (CH₂Cl₂–MeOH 95 : 5); [α]²_D²ϩ83.8 (c 1.0 in

CHCl₃); ν_max(KBr)/cm^Ϫ13445m, 2934m, 1711s, 1623s, 1382m,

1266m, 1194m, 1151s, 1088s, 1061m, 1040m; δ_H(300 MHz;

CDCl₃) 1.17 (3 H, d, J 6.8, 4-Me), 3.27 (1 H, m, 5-H), 3.39–3.50

(4 H, m, 6-H_a, 5-OMe), 3.57–3.75 (7 H, m, 6-H_b, 1-OMe,

3-OMe), 4.09 (1 H, dq, J 7.0 and 7.0, 4-H), 5.03 (1 H, s, 2-H);

δ_C(100.6 MHz; CDCl₃) 14.5 (4-Me), 36.0 (C-4), 51.3 (1-OMe),

55.6, 58.1 (3-OMe, 5-OMe), 61.4 (C-6), 83.6 (C-5), 91.5 (C-2),

168.7 (C-1), 176.5 (C-3); m/z (DCI) 236 (M ϩ NH₄^ϩ, 100%),

204 (100); HRMS found (M ϩ NH₄^ϩ) 236.1549; C₁₀H₁₈O₅

requires 236.1498.

residue by preparative TLC (CH₂Cl₂–MeOH 75 : 25) gave 22

ϩ

(20.2 mg, 51%) as a colourless oil; m/z (DCI) 388 (M ϩ NH₄

,

30%), 127 (30), 110 (100); HRMS found (M ϩ NH₄^ϩ) 388.1185;

C₁₅H₂₂N₂O₃S₂Si requires 388.1211.

Methyl (2E,4R,5R)-6-(tert-butyldimethylsilyloxy)-3,5-

dimethoxy-4-methylhex-2-enoate 23

Compound 19 (72.4 mg, 0.332 mmol) was treated with tert-

butyldimethylsilyl chloride and imidazole as described in the

synthesis of 20. The resulting product was puriﬁed by column

chromatography (cyclohexane–diethyl ether 6 : 1) to yield 23

(90.0 mg, 82%) as a colourless oil; R_f0.34 (cyclohexane–diethyl

ether 5 : 1); [α]²_D²ϩ56.5 (c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ12954s,

2929s, 2857m, 1716s, 1624s, 1472m, 1382m, 1257m, 1141s,

1100m, 837s, 778m; δ_H(300 MHz; CDCl₃) 0.03 [6 H, s,

Si(CH₃)₂], 0.88 [9 H, s, C(CH₃)₃], 1.15 (3 H, d, J 7.0, 4-Me), 3.34

(1 H, ddd, J 3.2, 6.8 and 8.1, 5-H), 3.47 (3 H, s, 5-OMe), 3.54

(1 H, dd, J 6.8 and 11.1, 6-H_a), 3.59–3.68 (7 H, m, 6-H_b, 1-OMe,

3-OMe), 4.01 (1 H, dq, J 7.0 and 8.1, 4-H), 4.97 (1 H, s, 2-H);

δ_C(100.6 MHz; CDCl₃) Ϫ5.4, Ϫ5.3 [Si(CH₃)₂], 14.6 (4-Me),

18.4 [C(CH₃)₃], 25.9 [C(CH₃)₃], 36.6 (C-4), 50.8 (1-OMe), 55.5,

59.4 (3-OMe, 5-OMe), 65.0 (C-6), 84.3 (C-5), 90.8 (C-2), 167.5

(C-1), 177.2 (C-3); m/z (DCI) 350 (M ϩ NH₄^ϩ, 100%); HRMS

found (M ϩ NH₄^ϩ) 350.2399; C₁₆H₃₂O₅Si requires 350.2363.

2Ј-[(1RS )-1-(tert-Butyldimethylsilyloxy)ethyl]-4-(tert-butyl-

dimethylsilyloxymethyl)-2,4Ј-bi-1,3-thiazole 20

Compound 18 (37.6 mg, 0.155 mmol) was dissolved in

anhydrous DMF (1 ml) containing tert-butyldimethylsilyl

chloride (150 mg, 1.0 mmol) and imidazole (68 mg, 1.0 mmol).

After stirring under a N₂atmosphere for 48 h at room temper-

ature, the reaction mixture was diluted with water and extracted

three times with diethyl ether. The combined organic layers

were washed with brine, dried over MgSO₄, and concentrated

in vacuo. Puriﬁcation of the residue by preparative TLC

(petroleum ether–ethyl acetate 10 : 1) gave 20 (62.3 mg, 85%) as

a colourless oil; R_f0.54 (petroleum ether–ethyl acetate 10 : 1);

ν_max(KBr)/cm^Ϫ12955s, 2929s, 2885m, 2857s, 1472m, 1462m,

1258m, 1178m, 1102s, 939m, 836s, 813m, 778s; δ_H(400 MHz;

CDCl₃) 0.11, 0.12, 0.13, 0.14 [12 H, 4 × s, 2 × Si(CH₃)₂], 0.96,

0.96 [18 H, 2 × s, 2 × C(CH₃)₃], 1.60 (3 H, d, J 6.6, CHCH₃),

4.90 (2 H, s, CH₂O), 5.12 (1 H, q, J 6.0, CHCH₃), 7.19, 7.80

(2 H, 2 × s, 2 × SCH); δ_C(100.6 MHz; CDCl₃) Ϫ5.3, Ϫ5.0, Ϫ4.6

[2 × Si(CH₃)₂], 18.1, 18.4 [2 × C(CH₃)₃], 25.4 (CHCH₃), 25.8,

26.0 [2 × C(CH₃)₃], 62.4 (CH₂O), 69.5 (CHCH₃), 113.9, 115.3

(2 × SCH), 149.4, 158.3, 162.9 178.9 (4 × C); m/z (DCI) 471

(M ϩ H^ϩ, 100%); HRMS found (M ϩ H^ϩ) 471.1990; C₂₁H₃₈-

N₂O₂S₂Si₂requires 471.1992.

(2E,4R,5R)-6-(tert-Butyldimethylsilyloxy)-3,5-dimethoxy-4-

methylhex-2-en-1-ol 24

To a solution of compound 23 (165 mg, 0.50 mmol) in

anhydrous THF (5 ml) was added under a N₂atmosphere at

Ϫ78 ЊC a 1 M solution of DIBAL-H in CH₂Cl₂(1 ml). After

stirring for 45 min at this temperature, the reaction was

quenched with a saturated. aqueous solution of potassium

sodium tartrate (5 ml). The reaction mixture was then treated

with ethyl acetate (5 ml) and stirred vigorously for 1.5 h at room

temperature. The aqueous layer was extracted three times with

ethyl acetate, the combined organic layers were washed with

brine, dried over MgSO₄, and concentrated in vacuo. Puriﬁ-

cation of the residue by column chromatography (heptane–

ethyl acetate 5 : 1, 3% triethylamine) gave 24 (107 mg, 71%) as a

colourless oil; R_f0.33 (heptane–ethyl acetate 2 : 1); [α]²_D²ϩ27.6

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13467m, 2953s, 2930s, 2857m,

1656m, 1463m, 1253m, 1142m, 1100s, 837s, 776m; δ_H(400

MHz; CDCl₃) 0.06, 0.07 [6 H, 2 × s, Si(CH₃)₂], 0.88 [9 H, s,

C(CH₃)₃], 1.12 (3 H, d, J 6.8, 4-Me), 3.01 (1 H, dq, J 6.8 and 6.8,

4-H), 3.14 (1 H, ddd, J 2.6, 4.6 and 7.9, 5-H), 3.41–3.51 (7 H, m,

6-H_a, 3-OMe, 5-OMe), 3.78 (1 H, dd, J 2.5 and 11.7, 6-H_b), 3.91

(1 H, m, 1-H_a), 4.15 (1 H, dd, J 8.9 and 12.0, 1-H_b), 4.84 (1 H,

dd, J 8.4 and 8.4, 2-H); δ_C(100.6 MHz; CDCl₃) Ϫ5.4, Ϫ5.1

[Si(CH₃)₂], 15.1 (4-Me), 18.6 [C(CH₃)₃], 26.0 [C(CH₃)₃], 35.3

{2Ј-[(1RS )-1-(tert-Butyldimethylsilyloxy)ethyl]-2,4Ј-bi-1,3-

thiazol-4-yl}methanol 21

Compound 20 (60.6 mg, 0.129 mmol) was dissolved in CH₂Cl₂

(1 ml) and MeOH (0.5 ml). A solution of camphorsulfonic acid

(6.0 mg, 0.025 mmol) in MeOH (0.5 ml) was added at 0 ЊC and

the reaction mixture was stirred for 3 h at room temperature.

After neutralization with triethylamine, the solvent was evapor-

ated and the residue was puriﬁed by preparative TLC (CH₂Cl₂–

MeOH 90 : 10) to give 21 (38.2 mg, 83%) as a colourless oil; R_f

0.50 (CH₂Cl₂–MeOH 95 : 5); ν_max(KBr)/cm^Ϫ13320m, 3111m,

2957s, 2856m, 1471m, 1431m, 1334m, 1257m, 1182s, 1110s,

1057m, 1023m, 943s, 835s, 773s, 814m, 678m; δ_H(300 MHz;

CDCl₃) 0.11, 0.14 [6 H, 2 × s, Si(CH₃)₂], 0.96 [9 H, s, C(CH₃)₃],

1.60 (3 H, d, J 6.4, CHCH₃), 4.81 (2 H, s, CH₂O), 5.17 (1 H, q,

J 6.2, CHCH₃), 7.19, 7.86 (2 H, 2 × s, 2 × SCH); δ_C(100.6 MHz;

CDCl₃) Ϫ5.0, Ϫ4.6 [Si(CH₃)₂], 18.1 [C(CH₃)₃], 25.1 (CHCH₃),

25.4 [C(CH₃)₃], 61.1 (CH₂O), 69.5 (CHCH₃), 115.1, 115.9

2498

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

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(C-4), 54.2, 58.8 (3-OMe, 5-OMe), 57.6 (C-1), 62.7 (C-6), 84.7

then 1 : 1) to give 27 (1.6 mg, 8%), 28 (7.0 mg, 41%), and 29

(6.3 mg, 44%) as colourless oils.

ϩ

(C-5), 97.6 (C-2), 162.1 (C-3); m/z (DCI) 322 (M ϩ NH₄

,

100%), 304 (70), 292 (50); HRMS found (M ϩ NH₄^ϩ) 322.2431;

28. Puriﬁcation by preparative HPLC (column B, eluent:

heptane–ethyl acetate 50 : 1). Analytical HPLC: R_t11.6 min

(column B, eluent: heptane–ethyl acetate 50 : 1); R_f0.23 (petrol-

eum ether–ethyl acetate 15 : 1); [α]²_D²ϩ17.9 (c 1.0 in CHCl₃);

ν_max(KBr)/cm^Ϫ12956s, 2929s, 2885m, 2858s, 1472m, 1257s,

1086s, 1072s, 836s, 776s; δ_C(100.6 MHz; CDCl₃) 11.3 (7-Me),

18.1, 18.2, 18.3, 18.5 [4 × C(CH₃)₃], 31.3 (C-9), 35.1 (C-5), 39.7

(C-7), 62.2 (C-10), 65.0 (C-1), 71.1, 73.1, 74.2, 77.0 (C-3, C-4,

C-6, C-8), 76.6 (C-2); m/z (DCI) 724 (M ϩ NH₄^ϩ, 100%);

HRMS found (M ϩ NH₄^ϩ) 724.5230; C₃₅H₇₈O₆Si₄requires

724.5219.

29. R_f0.10 (petroleum ether–ethyl acetate 15 : 1); ν_max(KBr)/

cm^Ϫ13411m, 2956s, 2929s, 2885m, 2858s, 1473m, 1256s, 1071s,

836s, 776s; δ_C(100.6 MHz; CDCl₃) Ϫ4.9, Ϫ4.8, Ϫ4.5, Ϫ4.4,

Ϫ4.3, Ϫ4.2 [3 × Si(CH₃)₂], 18.1, 18.2, 18.3 [3 × C(CH₃)₃], 25.8–

26.1 [C(CH₃)₃], 11.3 (7-Me), 30.6 (C-9), 35.1 (C-5), 39.7 (C-7),

62.0 (C-10), 63.8 (C-1), 70.8 (C-8), 73.8, 74.6 (C-2, C-3), 74.7

(C-6), 78.2 (C-4); m/z (DCI) 610 (M ϩ NH₄^ϩ, 100%), 593

[M ϩ H^ϩ, 40]; HRMS found (M ϩ NH₄^ϩ) 610.4386; C₂₉H₆₄-

O₆Si₃requires 610.4355.

C₁₅H₃₂O₄Si requires 322.2414.

Fragmentation of sorangicin A

Sorangicin A (3) (100 mg, 0.124 mmol) was dissolved in

anhydrous pyridine (2 ml) and treated at 0 ЊC with acetic

anhydride (2 ml) and a catalytic amount of DMAP. After

stirring for 18 h at room temperature, the reaction mixture

was concentrated, the residue dissolved in MeOH (5 ml) and

H₂O (0.1 ml), then stirred for 30 min at room temperature.

The solvent was removed in vacuo and the residue puriﬁed by

preparative HPLC (column A, eluent: MeOH–H₂O 80 : 20)

to give 21,22,25-tri-O-acetylsorangicin A⁴⁴(48.7 mg, 42%) as

a colourless oil. 21,22,25-Tri-O-acetylsorangicin A (35.4 mg,

0.038 mmol) was dissolved in CH₂Cl₂(440 µl) and methanol

(1.3 ml), and ozone was introduced for 5 min at Ϫ65 ЊC.

Dimethyl sulﬁde (150 µl) was added and the reaction mixture

was stirred for 45 min at room temperature, then evaporated

in vacuo. The residue was then dissolved in MeOH (10 ml) and

treated with polymer-supported borohydride (0.5 g). After

stirring for 3 h at room temperature, the reaction mixture was

ﬁltered and the solvent removed in vacuo. Puriﬁcation of

the residue by column chromatography (CH₂Cl₂–MeOH 95 : 5,

then 90 : 10) gave 25 (11.3 mg, 79%) and 26 (6.0 mg, 84%) as

colourless oils.

25. R_f0.46 (CH₂Cl₂–MeOH 90 : 10); m/z (DCI) 394 (M ϩ

NH₄^ϩ, 100%); HRMS found (M ϩ NH₄^ϩ) 394.2046; C₁₇H₂₈O₈

requires 394.2077.

26. R_f0.30 (CH₂Cl₂–MeOH 90 : 10); δ_H(300 MHz; CD₃OD)

0.95 (3 H, d, J 6.8, 6-Me), 1.63 (1 H, m, 6-H), 1.88 (1 H, dd,

J 1.5 and 11.7, 4-H_a), 2.05 (1 H, ddd, J 2.6, 6.8 and 11.7, 4-H_b),

3.48–3.62 (2 H, m, 7-H, 8-H_a), 3.68 (1 H, dd, J 1.9 and 11.3,

8-H_b), 3.82–3.93 (2 H, m, 1-H₂), 3.98 (1 H, m, 2-H), 4.23 (1 H,

m, 5-H), 4.40 (1 H, m, 3-H); δ_C(100.6 MHz; CD₃OD) 16.1

Synthesis of 30 by silylation of diol 29

Compound 29 (74.3 mg, 0.125 mmol) was dissolved in

anhydrous CH₂Cl₂(200 µl) and triethylamine (20 µl, 0.14

mmol), tert-butyldimethylsilyl chloride (20 mg, 0.132 mmol),

and a catalytic amount of DMAP were added at 0 ЊC. After

stirring for 22 h at room temperature, the reaction mixture was

diluted with water and extracted three times with CH₂Cl₂. The

combined organic layers were washed with brine, dried over

MgSO₄, and concentrated in vacuo. Puriﬁcation of the residue

by column chromatography (petroleum ether/diethyl ether

50 : 1, then petroleum ether–ethyl acetate 20 : 1, then 1 : 1) gave

27 (13.1 mg, 13%), 30 (28.2 mg, 38%), and 29 (28.2 mg, 38%) as

colourless oils.

30. Puriﬁcation by preparative HPLC (column B, eluent:

heptane–ethyl acetate 50 : 1).

(6-Me), 37.9 (C-6), 39.1 (C-4), 60.8 (C-1), 64.5 (C-8), 75.4 (C-3),

ϩ

80.3 (C-7), 80.7 (C-5), 84.8 (C-2); m/z (DCI) 206 (M ϩ NH₄

,

100%); HRMS found (M ϩ NH₄^ϩ) 206.1453; C₉H₁₆O₄requires

Analytical HPLC: R_t9.1 min (column B, eluent: heptane–

ethyl acetate 50 : 1); R_f0.28 (petroleum ether–ethyl acetate

15 : 1); [α]²_D²ϩ19.0 (c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ12956s,

2929s, 2885m, 2858s, 1472m, 1256s, 1072s, 836s, 776s; δ_C(100.6

MHz; CDCl₃) 11.2 (7-Me), 30.9 (C-9), 36.3 (C-5), 39.4 (C-7),

60.2 (C-10), 63.9 (C-1), 71.0, 71.0, 73.2, 73.9, 79.0 (C-2, C-3,

C-4, C-6, C-8); m/z (DCI) 724 (M ϩ NH₄^ϩ, 100%); HRMS

found (M ϩ NH₄^ϩ) 724.5221; C₃₅H₇₈O₆Si₄requires 724.5219.

206.1392.

Deacetylation of 25 and silylation to compound 27

Compound 25 (85.9 mg, 0.228 mmol) was dissolved in

anhydrous MeOH (9.7 ml) and treated with NaOMe (52 mg,

0.96 mmol). After stirring for 24 h at room temperature, the

reaction mixture was neutralized with Dowex 50 W X 8 (H^ϩ)

ion exchange resin, the reaction mixture was ﬁltered and the

solvent was removed in vacuo. The residue was dissolved in

anhydrous DMF (3.2 ml) containing tert-butyldimethylsilyl

chloride (970 mg, 6.43 mmol) and imidazole (436 mg, 6.40

mmol). After stirring under a N₂atmosphere for 72 h at room

temperature, the reaction mixture was diluted with water and

extracted three times with diethyl ether. The combined organic

layers were washed with brine, dried over MgSO₄, and concen-

trated in vacuo. Puriﬁcation of the residue by column chrom-

atography (petroleum ether–diethyl ether 50 : 1) gave 27 (109.1

mg, 58%) as a colourless oil; R_f0.35 (petroleum ether–diethyl

ether 50 : 1); ν_max(KBr)/cm^Ϫ12956s, 2929s, 2885m, 2858s,

1472m, 1257s, 1087s, 836s, 775s; δ_C(100.6 MHz; CDCl₃) 11.2

(7-Me), 31.1 (C-9), 36.7 (C-5), 39.4 (C-7), 61.0 (C-10), 65.2

(C-1), 70.7 (C-6), 71.3, 72.5, 77.8 (C-3, C-4, C-8), 76.3 (C-2);

m/z (ESI) 843.5 (M ϩ Na^ϩ, 100%).

Synthesis of 31 by silylation of diol 26

Compound 26 (117 mg, 0.62 mmol) was dissolved under a N₂

atmosphere in anhydrous DMF (1.9 ml) and anhydrous CH₂Cl₂

(9.6 ml). Anhydrous triethylamine (347 µl, 2.47 mmol) and

tert-butyldimethylsilyl triﬂuoromethanesulfonate (434 µl, 1.89

mmol) were then added at 0 ЊC. After stirring for 1 h at room

temperature, saturated aqueous sodium hydrogen carbonate

was added and the aqueous phase was extracted three times

with CH₂Cl₂. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. Puriﬁcation of the residue

by column chromatography (petroleum ether–ethyl acetate

20 : 1, then 10 : 1) gave 31 (148 mg, 57%) as a colourless oil;

R_f0.36 (petroleum ether–ethyl acetate 10 : 1); ν_max(KBr)/cm^Ϫ1

2956s, 2930s, 2882m, 2857s, 1472m, 1256s, 1130s, 1097s, 1057m,

1016m, 838s, 777s; δ_H(400 MHz; CDCl₃) 0.89, 0.90 [18 H, 2 × s,

2 × C(CH₃)₃], 0.91 (3 H, d, J 7.1, 6-Me), 1.60 (1 H, m, 6-H), 1.77

(1 H, dd, J 1.0 and 11.2, 4-H_a), 1.94 (1 H, ddd, J 2.5, 6.6 and

11.7, 4-H_b), 3.47 (1 H, m, 7-H), 3.59 (1 H, dd, J 4.1 and 11.2,

8-H_a), 3.67 (1 H, dd, J 3.0 and 11.2, 8-H_b), 3.82–3.94 (3 H, m,

1-H₂, 2-H), 4.18 (1 H, d, J 6.6, 5-H), 4.33 (1 H, br s, 3-H);

δ_C(100.6 MHz; CDCl₃) Ϫ5.2, Ϫ5.1 [Si(CH₃)₂], 16.0 (6-Me),

18.5 [C(CH₃)₃], 26.0 [C(CH₃)₃], 37.1 (C-6), 38.1 (C-4), 61.2

Synthesis of 28 and 29 by CSA-desilylation of 27

Compound 27 (19.7 mg, 0.024 mmol) was treated with cam-

phorsulfonic acid as described in the synthesis of 21 and the

residue was puriﬁed by column chromatography (petroleum

ether–diethyl ether 50 : 1, petroleum ether–ethyl acetate 20 : 1,

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

2499

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(C-1), 65.1 (C-8), 74.0 (C-3), 79.0 (C-7), 79.3 (C-5), 83.7 (C-2);

Compound 36 (111.2 mg, 0.252 mmol) was dissolved in

MeOH (6.3 ml) and treated with a solution of NaIO₄(80.7 mg,

0.377 mmol) in H₂O (6.3 ml). After stirring for 45 min at room

temperature, the organic solvent was removed in vacuo, brine

was added, and the reaction mixture was extracted three times

with ethyl acetate. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. The residue was dissolved in

MeOH (15 ml) and treated with polymer-supported boro-

hydride (0.76 g). After stirring for 3 h at room temperature, the

reaction mixture was ﬁltered and the solvent removed in vacuo.

Isolation by preparative HPLC (column A, eluent: MeOH–

H₂O 50 : 50) gave 37 (28.0 mg, 48%) and 38 (29.4 mg, 55%) as

colourless oils.

ϩ

m/z (DCI) 434 (M ϩ NH₄^ϩ, 100%); HRMS found (M ϩ NH₄

)

434.3151; C₂₁H₄₄O₄Si₂requires 434.3122.

Synthesis of 32 and 33 by CSA-desilylation of 31

Compound 31 (238 mg, 0.57 mmol) was treated with cam-

phorsulfonic acid as described in the synthesis of 21 and the

residue was puriﬁed by column chromatography (petroleum

ether–ethyl acetate 3 : 1, then ethyl acetate–MeOH 5 : 1) to give

31 (71.1 mg, 30%), a 1 : 1 mixture of 32 and 33 (72.2 mg, 42%),

and 26 (16.6 mg, 15%). Compounds 32 and 33 were separated

by column chromatography (hexane–ethyl acetate 3 : 2).

32. R_f0.13 (petroleum ether–ethyl acetate 2 : 1); [α]²_D²Ϫ68.4

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13433m, 2957s, 2930s, 2882m,

2858m, 1256m, 1129s, 1098m, 1055m, 837s, 777s; δ_H(300 MHz;

CDCl₃) 0.04, 0.05 (6 H, 2 × s, Si(CH₃)₂], 0.89 [9 H, s, C(CH₃)₃],

0.92 (3 H, d, J 6.8, 6-Me), 1.66 (1 H, m, 6-H), 1.80 (1 H, dd,

J 1.7 and 11.7, 4-H_a), 1.97 (1 H, ddd, J 2.8, 6.6 and 11.7, 4-H_b),

3.53 (1 H, m, 7-H), 3.58 (1 H, dd, J 4.1 and 10.9, 8-H_a), 3.67

(1 H, dd, J 2.6 and 10.9, 8-H_b), 3.82–4.02 (3 H, m, 1-H₂, 2-H),

4.22 (1 H, d, J 6.4, 5-H), 4.37 (1 H, m, 3-H); δ_C(100.6 MHz;

CDCl₃) Ϫ5.3, Ϫ5.1 [Si(CH₃)₂], 16.1 (6-Me), 18.4 [C(CH₃)₃],

26.0 [C(CH₃)₃], 36.9 (C-6), 38.2 (C-4), 61.3 (C-1), 64.8 (C-8),

74.1 (C-3), 79.2 (C-7), 79.5 (C-5), 82.8 (C-2); m/z (DCI) 320

(M ϩ NH₄^ϩ, 100%); HRMS found (M ϩ NH₄^ϩ) 320.2231;

C₁₅H₃₀O₄Si requires 320.2257.

37. Analytical HPLC: R_t10.8 min (column A, eluent:

MeOH–H₂O 50 : 50); ν_max(KBr)/cm^Ϫ13422s, 2968s, 2935s,

ϩ

,

2875m, 1703s, 1466m, 1011m; m/z (DCI) 248 (M ϩ NH₄

50%), 148 (100).

38. Analytical HPLC: R_t5.2 min (column A, eluent: MeOH–

H₂O 50 : 50); R_f0.62 (CH₂Cl₂–MeOH 85 : 15); [α]²_D²Ϫ25.7 (c 1.0

in CHCl₃); ν_max(KBr)/cm^Ϫ13377s, 2946m, 1508m, 1441m,

1190m, 1057s; δ_H(300 MHz; CDCl₃) 1.86 (2 H, m, 2-H₂), 1.99

(3 H, d, J 1.1, 4-Me), 2.68 (3 H, s, 8-Me), 3.82 (2 H, m, 1-H₂),

4.40 (1 H, t, J 6.0, 3-H), 6.59 (1 H, s, 5-H), 6.92 (1 H, s, 7-H);

δ_C(100.6 MHz; CDCl₃) 14.7 (4-Me), 19.1 (8-Me), 36.7 (C-2),

61.4 (C-1), 77.1 (C-3), 115.5 (C-7), 118.4 (C-5), 142.2 (C-4),

152.7 (C-6), 164.9 (C-8); m/z (DCI) 214 (M ϩ H^ϩ, 100%);

HRMS found (M ϩ H^ϩ) 214.0892; C₁₀H₁₅NO₂S requires

214.0902.

33. R_f0.29 (petroleum ether–ethyl acetate 2 : 1); [α]²_D²Ϫ76.2

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ13443m, 2956s, 2930s, 2882m,

2857m, 1257m, 1091s, 838s, 778s; δ_H(300 MHz; CDCl₃) 1.60

(1 H, m, 6-H), 1.77 (1 H, dd, J 1.5 and 11.7, 4-H_a), 2.00 (1 H,

ddd, J 2.6, 6.6 and 11.7, H-4_b), 3.46 (1 H, m, 8-H_a), 3.56 (1 H,

m, 7-H), 3.68 (1 H, m, 8-H_b), 3.86–3.94 (3 H, m, 1-H₂, 2-H),

4.21 (1 H, d, J 6.6, 5-H), 4.38 (1 H, m, 3-H); δ_C(100.6 MHz;

CDCl₃) Ϫ5.3, Ϫ5.2 [Si(CH₃)₂], 15.8 (6-Me), 18.4 [C(CH₃)₃],

26.0 [C(CH₃)₃], 36.7 (C-6), 38.3 (C-4), 60.8 (C-1), 63.9 (C-8),

74.1 (C-3), 78.4 (C-7), 78.9 (C-5), 83.5 (C-2); m/z (DCI) 320

(M ϩ NH₄^ϩ); HRMS found (M ϩ NH₄^ϩ, 100%) 320.2226;

C₁₅H₃₀O₄Si requires 320.2257.

(4R,5S,6S )-5,10-Bis(tert-butyldimethylsilyloxy)-2,4,6-

trimethyldecan-3-one 39

Compound 37 (25.7 mg, 0.112 mmol) was dissolved under a

N₂atmosphere in anhydrous CH₂Cl₂(2.2 ml) and at 0 ЊC

were added anhydrous triethylamine (63 µl, 0.44 mmol) and

tert-butyldimethylsilyl triﬂuoromethanesulfonate (77 µl, 0.33

mmol). After stirring for 1 h at room temperature, saturated

aqueous sodium hydrogen carbonate was added at 0 ЊC, and the

aqueous phase was extracted three times with CH₂Cl₂. The

combined organic layers were dried over MgSO₄and concen-

trated. Puriﬁcation of the residue by column chromatography

(petroleum ether–ethyl acetate 20 : 1) gave 39 (36.6 mg, 71%) as

a colourless oil; R_f0.73 (petroleum ether–ethyl acetate 10 : 1);

[α]²_D²Ϫ20.1 (c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ12957s, 2930s,

2858m, 1710m, 1472m, 1256m, 1101m, 1005m, 837s, 774s;

δ_H(300 MHz; CDCl₃) 0.88, 0.89 [18 H, 2 × s, 2 × C(CH₃)₃], 0.89

(3 H, d, J 7.0, 6-Me), 1.04, 1.06, 1.08 (9 H, 3 × d, 4-Me, 2-Me,

1-H₃), 2.75 (1 H, qqd, J 6.6 and 7.1, 2-H), 2.88 (1 H, dq, J 7.1

and 7.1, 4-H), 3.58 (2 H, t, J 6.6, 10-H₂), 3.83 (1 H, dd, J 3.3

and 6.6, 5-H); δ_C(100.6 MHz; CDCl₃) Ϫ5.2, Ϫ4.0, Ϫ3.9

[2 × Si(CH₃)₂], 18.4 [2 × C(CH₃)₃], 26.1, 26.0 [2 × C(CH₃)₃],

14.7 (4-Me), 16.6 (6-Me), 18.4, 18.8 (2-Me, C-1), 23.8 (C-8),

Fragmentation of epothilone A

Epothilone A (4) (670 mg, 1.36 mmol) was dissolved in MeOH

(16.8 ml), a 1 M aqueous solution of NaOH (16.8 ml) was

added, and the reaction mixture was stirred for 5 min at room

temperature. After addition of a 1 M aqueous solution of

NaH₂PO₄, the organic solvent was removed in vacuo and the

aqueous layer extracted four times with ethyl acetate. The com-

bined organic layers were washed with brine, dried over MgSO₄,

and concentrated in vacuo. Puriﬁcation of the residue by col-

umn chromatography (CH₂Cl₂–MeOH 95 : 5, then 80 : 20) pro-

vided 34 (141 mg, 24%) and 35 (451 mg, 65%) as colourless oils.

34. R_f0.68 (CH₂Cl₂–MeOH 85 : 15); m/z (DCI) 424 (M ϩ

H^ϩ, 100%), 324 (20); HRMS found (M ϩ H^ϩ) 424.2506;

C₂₃H₃₇NO₄S requires 424.2522.

31.7, 33.1 (C-7, C-9), 39.0 (C-6), 40.4 (C-2), 47.6 (C-4), 63.2

ϩ

(C-10), 76.6 (C-5), 217.3 (C-3); m/z (DCI) 476 (M ϩ NH₄

,

100%); HRMS found (M ϩ NH₄^ϩ) 476.3934; C₂₅H₅₄O₃Si₂

35. R_f0.44 (CH₂Cl₂–MeOH 85 : 15); m/z (DCI) 512 (M ϩ

H^ϩ, 10%), 424 (30), 324 (100), 206 (90).

requires 476.3955.

(4R,5S,6S )-5,10-Dihydroxy-2,4,6-trimethyldecan-3-one 37 and

(3S,4E )-4-methyl-5-(2-methyl-1,3-thiazol-4-yl)pent-4-ene-1,3-

diol 38

(4R,5S,6S )-5-(tert-Butyldimethylsilyloxy)-10-hydroxy-2,4,6-

trimethyldecan-3-one 40

Compound 39 (47.2 mg, 0.103 mmol) was treated with cam-

phorsulfonic acid as described in the synthesis of 21 and the

residue was puriﬁed by column chromatography (petroleum

ether–ethyl acetate 4 : 1) to give 40 (28.0 mg, 79%) as a colour-

less oil; R_f0.34 (petroleum ether–ethyl acetate 4 : 1); [α]²_D²Ϫ22.0

(c 1.0 in CHCl₃); ν_max(KBr)/cm^Ϫ12958s, 2933s, 2858m, 1708s,

1464m, 1257m, 1063m, 1005s, 837s, 774s; δ_H(300 MHz; CDCl₃)

0.88 [9 H, s, C(CH₃)₃], 0.90 (3 H, d, J 7.0, 6-Me), 1.05, 1.06, 1.08

(9 H, 3 × d, 4-Me, 2-Me, 1-H₃), 2.76 (1 H, qqd, J 6.8 and 7.0,

2-H), 2.88 (1 H, dq, J 7.0 and 7.0, 4-H), 3.61 (2 H, t, J 6.4,

10-H₂), 3.84 (1 H, dd, J 3.2 and 6.8, 5-H); δ_C(100.6 MHz;

Compound 34 (155 mg, 0.366 mmol) was dissolved in THF

(1.4 ml) and H₂O (1.4 ml). A 1 M aqueous solution of H₂SO₄

(730 µl) was added at 0 ЊC and the reaction mixture was stirred

for 5 h at room temperature. After addition of saturated aque-

ous sodium hydrogen carbonate, THF was removed in vacuo

and the aqueous layer extracted three times with ethyl acetate.

The combined organic layers were dried over MgSO₄and con-

centrated in vacuo. Puriﬁcation of the residue by column

chromatography (CH₂Cl₂–MeOH 95 : 5) gave diol 36 (111.2 mg,

69%) as a colourless oil; R_f0.55 (CH₂Cl₂–MeOH 85 : 15).

2500

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

View Article Online

CDCl₃) Ϫ4.0, Ϫ3.9 [Si(CH₃)₂], 18.4 [C(CH₃)₃], 26.1 [C(CH₃)₃],

was diluted with brine, extracted three times with ethyl acetate,

14.8 (4-Me), 16.5 (6-Me), 18.2, 18.8 (2-Me, C-1), 23.8 (C-8),

dried over MgSO₄, and concentrated in vacuo. The residue was

dissolved in tert-butanol (12.8 ml), 2-methylbut-2-ene (2.4 ml)

was added, then a solution of NaClO₂(207 mg, 2.29 mmol) and

NaH₂PO₄(158 mg) in H₂O (3 ml). The reaction mixture was

stirred for 1 h at room temperature, then a second portion of

NaClO₂(207 mg, 2.29 mmol) and NaH₂PO₄(158 mg) in H₂O

(3 ml) was added. After stirring for further 2 h at room tem-

perature, the solvent was removed in vacuo, the residue was

partitioned between ethyl acetate and brine, and the aqueous

layer extracted twice with ethyl acetate. The combined organic

layers were dried over MgSO₄and concentrated in vacuo. Puri-

ﬁcation of the residue by preparative HPLC (column A,

MeOH–H₂O, 50 mM NH₄OAc, pH 5.5) and subsequent extrac-

tion of product containing fractions after removal of MeOH at

pH 4 gave 45 (77.6 mg, 86%) and 46 (67.2 mg, 49%) as colour-

less oils.

31.7, 33.1 (C-7, C-9), 38.9 (C-6), 40.4 (C-2), 47.6 (C-4), 62.8

ϩ

(C-10), 76.6 (C-5), 217.5 (C-3); m/z (DCI) 362 (M ϩ NH₄

,

100%); HRMS found (M ϩ NH₄^ϩ) 362.3083; C₁₉H₄₀O₃Si

requires 362.3090.

(3S,4E )-1,3-Bis(tert-butyldimethylsilyloxy)-4-methyl-5-

(2-methyl-1,3-thiazol-4-yl)pent-4-ene 41

Compound 38 (19.6 mg, 0.092 mmol) was treated with tert-

butyldimethylsilyl triﬂuoromethanesulfonate as described in the

synthesis of 39. Puriﬁcation of the residue by preparative TLC

(petroleum ether–ethyl acetate 10 : 1) gave 41⁴⁷(34.5 mg, 85%)

as a colourless oil; R_f0.63 (petroleum ether–ethyl acetate 10 : 1);

[α]²_D²ϩ 4.1 (c 1.0 in CHCl₃); m/z (DCI) 442 (M ϩ H^ϩ, 100%);

HRMS found (M ϩ H^ϩ) 442.2609; C₂₂H₄₃NO₂SSi₂requires

442.2631.

45. R_f0.32 (CH₂Cl₂–MeOH 85 : 15); [α]²_D²ϩ2.5 (c 1.0 in

CHCl₃); [α]²_D²Ϫ18.7 (c 1.0 in MeOH); δ_HNMR (300 MHz;

CD₃OD) 1.34 (t, J 7.3, NCH₂CH₃), 2.05 (3 H, d, J 1.1, 4-Me),

2.50–2.66 (2 H, m, 2-H₂), 2.72 (3 H, s, 8-Me), 3.24 (q, J 7.3,

NCH₂CH₃), 4.60 (1 H, m, 3-H), 6.61 (1 H, s, 5-H), 7.23 (1 H, s,

7-H); δ_C(100.6 MHz; CD₃OD) 9.2 (NCH₂CH₃), 14.6 (4-Me),

18.6 (8-Me), 42.4 (C-2), 47.8 (NCH₂CH₃), 75.1 (C-3), 117.1

(C-7), 119.6 (C-5), 143.0 (C-4), 153.7 (C-6), 166.8 (C-8), 176.0

(C-1); m/z (DCI) 228 (M ϩ H^ϩ, 100%); HRMS found

(M ϩ H^ϩ) 228.0701; C₁₀H₁₃NO₃S requires 228.0694.

46. R_f0.58 (CH₂Cl₂–MeOH 85 : 15); δ_C(100.6 MHz;

CD₃OD) 12.5 (8-Me), 16.8 (6-Me), 20.4, 21.3 (2 × 4-Me), 23.8

(C-10), 32.2 (C-9), 37.3 (C-8), 38.7 (C-2, C-11), 44.1 (C-6), 52.2

(1-OMe), 53.8 (C-4), 73.6 (C-3), 76.8 (C-7), 174.3 (C-1, C-12),

221.1 (C-5); m/z (DCI) 364 (M ϩ NH₄^ϩ, 100%), 220 (45), 162

(40); HRMS found (M ϩ NH₄^ϩ) 364.2351; C₁₇H₃₀O₇requires

364.2335.

(3S,4E )-3-(tert-Butyldimethylsilyloxy)-4-methyl-5-(2-methyl-

1,3-thiazol-4-yl)pent-4-en-1-ol 42

Compound 41 (206.8 mg, 0.468 mmol) was treated with cam-

phorsulfonic acid as described in the synthesis of 21 and the

residue was puriﬁed by column chromatography (petroleum

ether–ethyl acetate 5 : 2, then 2 : 1) to give 42⁴³(134 mg, 88%) as

a colourless oil; R_f0.36 (petroleum ether–ethyl acetate 2 : 1);

[α]²_D²Ϫ32.9 (c 1.0 in CHCl₃); m/z (DCI) 328 (M ϩ H^ϩ, 100%);

HRMS found (M ϩ H^ϩ) 328.1759; C₁₆H₂₉NO₂SSi requires

328.1767.

(3S,4E )-4-Methyl-5-(2-methyl-1,3-thiazol-4-yl)pent-4-ene-1,3-

diol 38 and methyl (3S,6R,7R,8R)-3,7,12-trihydroxy-4,4,6,8-

tetramethyl-5-oxododecanoate 44

Compound 35 (111.8 mg, 0.218 mmol) was treated with a 1 M

aqueous solution of H₂SO₄as described in the synthesis of 36.

The crude product was dissolved in MeOH (2.1 ml) and toluene

(2.1 ml), and subsequently treated with a 2.0 M solution of

(trimethylsilyl)diazomethane (250 µl) in portions over 2 h. After

stirring for 0.5 h at room temperature, the reaction mixture was

concentrated in vacuo. Puriﬁcation of the residue by preparative

TLC (CH₂Cl₂–MeOH 85 : 15) gave 43 (63.8 mg, 54%) as a

colourless oil; R_f0.58 (CH₂Cl₂–MeOH 85 : 15); m/z (DCI) 544

(M ϩ H^ϩ, 10%), 422 (60), 342 (50), 220 (100).

Compound 43 (63.8 mg, 0.117 mmol) was subsequently

treated with NaIO₄and polymer-supported borohydride as

described for the synthesis of 37 and 38. Isolation by prepar-

ative HPLC (column A, eluent: MeOH–H₂O 50 : 50) gave 38

(19.6 mg, 49%) and 44 (17.7 mg, 41%) as colourless oils.

Methyl [(2S,4S,5R,6S )-4,5-bis(tert-butyldimethylsilyloxy)-6-

hydroxymethyltetrahydro-2H-pyran-2-yl] acetate 47

Ambruticin S (5) (106 mg, 0.216 mmol) was dissolved in

ethanol (8 ml) and treated with a freshly prepared solution of

diazomethane in diethyl ether. Excess diazomethane was des-

troyed by addition of acetic acid and the reaction mixture was

concentrated in vacuo. The residue was dissolved under a N₂

atmosphere in anhydrous CH₂Cl₂(4 ml) and treated at 0 ЊC with

triethylamine (120 µl, 0.87 mmol) and tert-butyldimethylsilyl

triﬂuoromethanesulfonate (150 µl, 0.65 mmol). After stirring

for 1 h at room temperature, the reaction mixture was diluted

with CH₂Cl₂and washed with saturated aqueous sodium

hydrogen carbonate. The organic phase was dried over MgSO₄

and concentrated in vacuo; R_f0.44 (hexane–ethyl acetate 9 : 1).

The crude product was dissolved in CH₂Cl₂(2.5 ml). To 1.0 ml

of this solution was added MeOH (3 ml), and ozone was intro-

duced for 3 min at Ϫ74 ЊC. The reaction mixture was evapor-

ated in vacuo, the residue dissolved in MeOH (4 ml) and CH₂Cl₂

(2.5 ml), and treated at 0 ЊC with sodium borohydride (40 mg).

After stirring for 1 h at room temperature, the reaction mixt-

ure was treated with a 1 M aqueous solution of NaH₂PO₄–

K₂HPO₄(pH 7) and extracted three times with CH₂Cl₂. The

combined organic layers were dried over MgSO₄and concen-

trated in vacuo. Puriﬁcation of the residue by column chrom-

atography (petroleum ether–ethyl acetate 9 : 2) gave 47 (14.3

mg, 40%) as a colourless oil; R_f0.67 (petroleum ether–ethyl

acetate 2 : 1); ν_max(KBr)/cm^Ϫ12955s, 2929s, 2857s, 1744s,

1473m, 1258m, 1115s, 837s, 779m; δ_H(300 MHz; CDCl₃) 0.88,

0.90 [18 H, 2 × s, 2 × C(CH₃)₃], 1.38 (1 H, m, 4-H_a), 1.99 (1 H,

ddd, J 1.1, 3.4, and 9.5, H-4_b), 2.41 (1 H, dd, J 3.8 and 11.4,

2-H_a), 2.56 (1 H, dd, J 5.7 and 11.4, 2-H_b), 3.22 (1 H, m, 7-H),

3.31 (1 H, dd, J 6.1 and 6.9, 6-H), 3.57 (1 H, m, 8-H_a), 3.65–3.72

(4 H, m, 5-H, 1-OMe), 3.78 (1 H, m, 8-H_b), 3.87 (1 H, m, 3-H);

δ_C(100.6 MHz; CDCl₃) Ϫ4.5, Ϫ4.1, Ϫ2.9, Ϫ2.8 [2 × Si(CH₃)₂],

44. Analytical HPLC: R_t8.5 min (column A, eluent: MeOH–

H₂O 50 : 50); ν_max(KBr)/cm^Ϫ13361m, 2940m, 1739s, 1688m,

1438m, 994m; δ_H(300 MHz; CDCl₃) 0.84 (3 H, d, J 6.8, 8-Me),

1.04 (3 H, d, J 7.0, 6-Me), 1.13, 1.19 (6 H, 2 × s, 4-Me), 2.37

(1 H, dd, J 10.0 and 16.2, 2-H_a), 2.46 (1 H, dd, J 2.8 and 16.2,

2-H_b), 3.63 (2 H, t, J 6.2, 12-H₂), 3.37 (1 H, m, 7-H), 3.25 (1 H,

m, 6-H), 3.71 (3 H, s, COOMe), 4.25 (1 H, m, 3-H); δ_C(100.6

MHz; CDCl₃) 10.0 (8-Me), 15.6 (6-Me), 18.9, 21.3 (2 × 4-Me),

22.7 (C-10), 32.3 (C-11), 33.0 (C-9), 35.4 (C-8), 36.4 (C-2), 40.9

(C-6), 52.1 (C-4, 1-OMe), 62.9 (C-12), 72.4 (C-3), 74.8 (C-7),

173.4 (C-1), 222.3 (C-5); m/z (DCI) 350 (M ϩ NH₄^ϩ, 100%),

ϩ

248 (30), 220 (50), 148 (40); HRMS found (M ϩ NH₄

)

350.2523; C₁₇H₃₂O₆requires 350.2543.

(3S,4E )-3-Hydroxy-4-methyl-5-(2-methyl-1,3-thiazol-4-yl)pent-

4-enoic acid 45 and (5R,6R,7R,10S )-6,10-dihydroxy-12-

methoxy-5,7,9,9-tetramethyl-8,12-dioxododecanoic acid 46

Compound 43 (215 mg, 0.40 mmol) was dissolved in MeOH

(9.9 ml) and treated with a solution of NaIO₄(126 mg, 0.56

mmol) in H₂O (9.9 ml). After stirring for 45 min at room tem-

perature, the MeOH was removed in vacuo, the aqueous layer

J. Chem. Soc., Perkin Trans. 1, 2002, 2490–2503

2501

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18.4, 18.1 [2 × C(CH₃)₃], 26.3, 26.1 [2 × C(CH₃)₃], 40.5 (C-2),

3

Coupling of amino acids. The protected amino acid

40.7 (C-4), 51.8 (1-OMe), 63.0 (C-8), 72.1 (C-3), 73.6 (C-6), 74.8

(0.3 M), 1-hydroxybenzotriazole (HOBt) (1.7 equiv.) and

N,N-diisopropylcarbodiimide (DIC) (1.3 equiv.) was dissolved

in NMP. After 30 min the mixture was spotted onto the mem-

brane (2 × 1.5 µl per spot, 5 equiv.) and the reaction was

allowed to proceed for 60 min. The membrane was washed with

DMF (3×) and DCM (3×).

ϩ

(C-5), 80.8 (C-7), 171.2 (C-1); m/z (DCI) 466 (M ϩ NH₄

,

100%); HRMS found (M ϩ NH₄^ϩ) 466.3020; C₂₉H₅₀O₆Si₂

requires 466.3008.

Fragmentation of apicularen A

4

Determination of membrane loading. A single spot was cut

Apicularen A (6) (10.7 mg, 0.024 mmol) was treated with tert-

butyldimethylsilyl chloride and imidazole as described in the

synthesis of 20. Puriﬁcation of the residue by column chrom-

atography (petroleum ether–ethyl acetate 9 : 2, 2% triethyl-

amine) gave 3,11-di-O-tert-butyldimethylsilylapicularen A (11.4

mg, 71%) as a colourless oil; R_f0.39 (petroleum ether–ethyl

acetate 4 : 1); m/z (DCI) 687 (M ϩ NH₄^ϩ, 100%); HRMS found

(M ϩ NH₄^ϩ) 687.4225; C₃₇H₅₉NO₆Si₂requires 687.4274.

out and placed in an Eppendorf vial and a solution of 20%

piperidine in DMF (1 ml) was added. After 30 min the absorb-

ance at 301 nm was measured and loading calculated using the

molar absorption coeﬃcient of the dibenzofulvene–piperidine

adduct (ε = 7800). The spot loading of the Phe–Phe–Amp

tripeptide was determined to be 170 nmol.

5

Boc-deprotection. The membrane was treated with 50%

TFA (2 × 45 min), then washed with a 5% solution of diiso-

propylethylamine (DIEA) in DMF (3×), then with DMF ( ×)

and DCM (3×).

3,11-Di-O-tert-butyldimethylsilylapicularen

A (11.4 mg,

0.017 mmol) was dissolved in CH₂Cl₂(0.5 ml) and methanol

(1.5 ml), and ozone was introduced for 5 min at Ϫ75 ЊC. Di-

methyl sulﬁde (200 µl) was added and the reaction mixture

was stirred for 45 min at room temperature, then concentrated

in vacuo. The residue was dissolved in MeOH (2 ml) and

CH₂Cl₂(1 ml), and treated at 0 ЊC with NaBH₄(5 mg). After

stirring for 2 h at room temperature, the reaction mixture was

partitioned between ethyl acetate and a 1 M aqueous solution

of NaH₂PO₄–K₂HPO₄(pH 7), and the aqueous layer extracted

twice with ethyl acetate. The combined organic layers were

washed with brine, dried over MgSO₄, and concentrated in

vacuo. Puriﬁcation of the residue by column chromatography

(petroleum ether–ethyl acetate 3 : 1, 1% triethylamine) gave 48

(7.5 mg, 80%) as a colourless oil; R_f0.51 (petroleum ether–ethyl

acetate 2 : 1); δ_H(300 MHz; CDCl₃) 0.89, 0.96 [18 H, 2 × s,

2 × C(CH₃)₃], 2.38 (1 H, dd, J 1.3 and 14.9, 8-H_a), 3.47 (1 H, dd,

J 10.2 and 14.9, 8-H_b), 3.93–4.08 (2 H, m, 9-H, 11-H), 4.31

(1 H, m, 13-H), 5.70 (1 H, m, 15-H), 6.68–6.74 (2 H, m, 4-H,

6-H), 7.11 (1 H, dd, J 7.9 and 7.9, 5-H); δ_C(75.5 MHz; CDCl₃)

Ϫ4.7, Ϫ4.1, Ϫ3.9 [2 × Si(CH₃)₂], 18.5, 18.1 [2 × C(CH₃)₃], 25.9

[2 × C(CH₃)₃], 37.5 (C-16), 38.5 (C-14), 39.1 (C-10), 39.3

(C-12), 39.5 (C-8), 59.1 (C-17), 65.6 (C-11), 66.2 (C-13), 72.1

(C-15), 73.9 (C-9), 117–3 (C-4), 123.2 (C-6), 128.1 (C-2), 129.6

(C-5), 139.5 (C-7), 151.9 (C-3), 169.9 (C-1); m/z (DCI) 568

(M ϩ NH₄^ϩ, 100%); HRMS found (M ϩ NH₄^ϩ) 568.3490;

C₂₉H₅₀O₆Si₂requires 568.3521.

6

Coupling of natural product fragments. The primary alco-

hol fragment (0.3 M), p-nitrophenyl chloroformate (0.9 equiv.)

and DMAP (1.0 equiv.) were dissolved in a 1 : 1 (v/v) mixture of

pyridine and NMP. After 30 min the precipitate was removed

by centrifugation and the solution was spotted onto the mem-

brane (3 × 1.3 µl per spot, 7–8 equiv.). The reaction was allowed

to proceed for 2 h and the membrane was washed with DMF

(3 ×) and DCM (3 ×). Coupling yields were found to be >80%.

7

TBS-deprotection. A solution of HF (70% in pyridine)

was diluted 1 : 1 with DMF (caution!) and cooled to room

temperature. The membrane was treated with the HF-solution

for 3 h and was then washed with DMF (3×) and DCM (3×).

8

Photolytic cleavage. A single spot was cut out and placed

in an Eppendorf vial. Cleavage from the membrane was per-

formed in MeOH by irradiating for 3 h with UV light at 365

nm. The average compound recovery by cleavage from the

linker was determined to be 80%. The product was analysed by

HPLC-MS.

Analysis was performed by reverse-phase HPLC ESI-MS

measured at 254 nm and 211 nm. For peak area integration the

diﬀerent molar absorption coeﬃcients of epothilone A (ε =

17800 l mol^Ϫ1cm^Ϫ1) and phenylalanine (ε = 11700 l mol^Ϫ1cm^Ϫ1

)

measured in MeOH at 211 nm were taken into account. At

254 nm all compounds to which the epothilone fragment 42 is

coupled give intensive signals whereas the absorption of the

phenylalanine residues at this wavelength is negligible. The

chromatogram measured at 211 nm reveals on the other

hand additionally impurities that derive from Phe–Phe-linked

intermediates, which contain no epothilone fragment.

Compound 49. ∼100 nmol (60% overall yield); R_t13.2 min

(linear gradient of 5–95% aqueous CH₃CN, 5 mM NH₄OAc,

pH 5.5 over 20 min); m/z (ESI) 877.0 (M ϩ H^ϩ); C₄₄H₅₆N₆O₁₁S

requires 877.4.

SPOT synthesis of a natural product-derived hybrid structure

General procedures. All steps were carried out with the mem-

brane placed in a polypropylene tray with a tight cover. Except

for spotting reactions, the membrane was covered with respect-

ive solvents and reagent solutions and gently agitated on a

rocker plate for the times indicated. Reaction conditions were

optimised with model compounds to reach best possible results

in coupling, deprotection, and cleavage reactions. The spot-size

for manual spot synthesis with a spot volume of 1.2 µl is ∼0.28

cm².

Acknowledgements

1

Coupling of the Fmoc-aminoethyl photolinker onto the

This work was supported by the Bundesministerium für

Bildung und Forschung (Leitprojekt Verbund “Validierte

Lead-Target-Systeme”). The authors thank C. Churt for her

skillfull assistance, Dr. R Jansen and N. Dankers for isolation

of sorangicin A and apicularen A, and H. Steinmetz and

I. Schleicher for providing myxothiazol A and epothilone A. We

further thank I. Schleicher and U. Nolte for HPLC-MS

analysis, C. Kakoschke and B. Jaschok-Kentner for recording

the NMR spectra, and R. Christ for measuring mass spectra.

APEG-membrane. 4-{4-[1-(Fmoc-amino)ethyl]-2-methoxy-5-

nitrophenoxy}butanoic acid (0.2 M), 1-hydroxybenzotriazole

(HOBt) (1.7 equiv.) and N,N-diisopropylcarbodiimide (DIC)

(1.3 equiv.) were dissolved in N-methylpyrrolidin-2-one (NMP).

After 30 min the solution was spotted onto the membrane

(2 × 1.2 µl per spot, 3 equiv.) and the reaction was allowed to

proceed for 60 min. The membrane was washed three times

with DMF and twice with ethanol. The remaining amino-

groups were then capped by acetylation with 2% acetic

anhydride in DMF overnight and the membrane was washed

again three times with DMF and twice with ethanol.

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2503

Article Doi

Natural product-derived building blocks for combinatorial synthesis. Part 1. Fragmentation of natural products from myxobacteria

DOI: 10.1039/b206953a

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