Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

Cyclopropyl Containing Fatty Acids as Mechanistic Probes for

Cytochromes P450

Max J. Cryle,^†Paul R. Ortiz de Montellano,^‡and James J. De Voss*^,†

Department of Chemistry, University of Queensland, St. Lucia, Brisbane, Australia 4072, and

Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280

j.devoss@uq.edu.au

Received November 13, 2004

The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense

debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds

(radical clocks), which can produce both unrearranged and ring opened products upon oxidation,

have been key tools in these investigations. In this study, we introduce several cyclopropyl containing

fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation.

The probes are shown to be capable of distinguishing radical from cationic intermediates due to

the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in

an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation

isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these

probes by P450_BM3and P450_BioIgives results consistent with a radical but not a cationic intermediate

in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened

products gives remarkably homogeneous rates for oxygen rebound of (2-3) × 10¹⁰s^-1. The effects

of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation

are discussed.

Introduction

mechanistic pathway involves hydrogen atom abstraction

by a high valent iron oxo species followed by a rebound

of the iron tethered hydroxyl radical onto the substrate

carbon radical to give the hydroxylated product.³Detec-

tion of the substrate radical intermediate predicted by

this mechanism provides powerful support for this path-

way. One approach, introduced in 1987 and subsequently

used extensively for this purpose, employs so-called

cyclopropyl radical clocks.⁴These compounds utilize the

strain inherent in the cyclopropyl ring system to report

the presence of a radical R to the ring via formation of

products arising from ring opening reactions. In addition

to simply providing evidence for the presence of a radical

intermediate, quantitation of both unrearranged and

ring-opened oxidation products, coupled with knowledge

of the rate of ring opening, allows the lifetime of the

radical intermediate to be calculated.

The cytochromes P450 (P450s) constitute a superfamily

of heme-dependent monooxygenases that catalyze a

variety of oxidative transformations, including among

others, the regio- and stereoselective hydroxylation of

aliphatic carbons and carbon-carbon bond cleavage.^1-3

Such reactions play an important role in mammalian

xenobiotic detoxification as well as in a wide variety of

prokaryotic and eukaryotic biosynthetic and biodegra-

dative pathways. The mechanism of P450 mediated

hydroxylation has thus been the subject of intensive

investigation for many years. Currently, the accepted

* Corresponding author. Fax: 61 7 3365 4299. Tel: 61 7 3365 3825.

^†University of Queensland.

^‡University of California, San Francisco.

(1) Ortiz de Montellano, P. R. In Cytochrome P450: Structure,

Mechanism, and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.;

Plenum Press: New York, 1995; p 245.

The results from such probes have been generally

consistent with the presence of a radical intermediate in

(2) Cryle, M. J.; Stok, J. E.; De Voss, J. J. Aus. J. Chem. 2003, 56,

749-762.

(3) Ortiz de Montellano, P. R.; De Voss, J. J. Nat. Prod. Rep. 2002,

19, 477-493.

(4) Ortiz de Montellano, P. R.; Stearns, R. A. J. Am. Chem. Soc.

1987, 109, 3415-20.

Published on Web 02/26/2005

J. Org. Chem. 2005, 70, 2455-2469

2455

Cryle et al.

the oxidation^4-7and thus are in harmony with a variety

of other experimentally observed features of P450 cata-

lyzed hydroxylations, such as allylic transposition and

stereochemical scrambling. However, as increasingly

sophisticated probes have been employed to characterize

more fully the nature and lifetime of the intermediate,

conflicting results have arisen.^8-12These include calcu-

lated lifetimes of radical intermediates more consistent

with a transition state and an effectively concerted

reaction, as well as indications of a cationic oxidation

pathway.^9,11

FIGURE 1. Cyclopropyl fatty acids 1a-4a and sites of

oxidation for which they would provide mechanistic informa-

tion.

Recent density functional theory calculations have

reconciled the conflicting results from such probes by

suggesting that a two state reactivity paradigm applies

to the reactive iron oxo species that mediates P450

oxidations.^13,14In this, the oxidation can be initiated by

one of two different electromers of the iron oxo interme-

diate, and the reaction then proceeds via two parallel H

atom abstraction pathways, one with a true radical

intermediate and one that is effectively concerted. Im-

portantly, these studies suggest that, in part, the nature

of the substrate may determine which of the two path-

ways predominates, raising the possibility that sophis-

ticated probes may reveal mechanistic characteristics of

their own oxidation and not of all P450 oxidation reac-

tions. In general, the probes employed to date have

previously been chosen for, or designed to possess, specific

properties, such as faster rates of ring opening or

differing ring scission pathways for radical versus cat-

ionic intermediates. We thus set out to synthesize a series

cyclopropyl radical clocks that, while still reporting on a

radical intermediate, were as close as possible to natural

P450 substrates.

Our interest in fatty acid metabolizing P450s has

led us to investigate P450_BM3(CYP102A1), P450_BioI

(CYP107H1), and CYP119, all of which possess interest-

ing properties. P450_BM3, a catalytically self-sufficient

P450 isolated from Bacillus megaterium, possesses one

of the fastest rates of turnover for any P450 and is also

very well coupled for the hydroxylation of fatty acids and

epoxidation of unsaturated acids.^15,16P450_BioI, encoded

within the biotin operon of B. subtilis,¹⁷is also capable

of nonterminal aliphatic hydroxylation.¹⁸Biosyntheti-

cally, however, P450_BioIperforms carbon-carbon bond

cleavage of an unactivated fatty acid chain via alcohol

and vicinal diol intermediates.^19,20The true enzymic

substrate is believed to be the acyl carrier protein bound,

rather than free, fatty acid. Finally, CYP119 is a P450

isolated from the thermophilic bacteria Sulfolobus sol-

fataricus that remains catalytically active at tempera-

tures in excess of 70 °C and is capable of high-

temperature hydroxylation of medium chain length fatty

acids.^21-23With the variety of properties that these

enzymes possess, it appeared that cyclopropyl radical

probes based upon a fatty acid template would be ideal

as they would allow us to determine if the mechanism of

oxidation in these very different P450s was the same or

different in each case. Thus, we report here the synthesis

and characterization of a number of novel cyclopropyl

fatty acids and demonstrate that they are also capable

of distinguishing radical from cationic intermediates. The

results of our investigations into the nature of the

reactive intermediate in the oxidations catalyzed by these

three enzymes employing these probes will also be

detailed. A preliminary report of the utility of some these

probes with P450_BM3has appeared.²⁴

Results and Discussion

Probe Design and Synthesis. For the purpose of this

study, all the probes were designed on a basic fatty acid

backbone to exploit ω-2 hydroxylation, which is observed

in straight chain fatty acids with P450_BM3, P450_BioI, and

CYP119 (Figure 1). As such, this allowed the develop-

ment of three probes for both C₁₄and C₁₆equivalent²⁵

series of fatty acid probes: these probes possess either a

terminal cyclopropyl ring or a Z or E internal cyclopropyl

ring. Such internal cyclopropyl probes would also be able

to exploit the ω-5 oxidation site with P450_BioIdue to the

poor regioselectivity of oxidation observed with this

(5) Auclair, K.; Hu, Z.; Little, D. M.; Ortiz de Montellano, P. R.;

Groves, J. T. J. Am. Chem. Soc. 2002, 124, 6020-6027.

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Am. Chem. Soc. 1992, 114, 10915-10921.

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(11) Toy, P. H.; Newcomb, M.; Hollenberg, P. F. J. Am. Chem. Soc.

1998, 120, 7719-7729.

(18) Cryle, M. J.; Matovic, N. J.; De Voss, J. J. Org. Lett. 2003, 5,

3341-3344.

(19) Stok, J. E.; De Voss, J. J. Arch. Biochem. Biophys. 2000, 384,

351-360.

(12) Atkinson, J. K.; Ingold, K. U. Biochemistry 1993, 32, 9209-

9214.

(20) Cryle, M. J.; De Voss, J. J. Chem. Commun. 2004, 86-87.

(21) Puchkaev, A. V.; Wakagi, T.; Ortiz de Montellano, P. R. J. Am.

Chem. Soc. 2002, 124, 12682-12683.

(13) Shaik, S.; de Visser, S. P.; Ogliaro, F.; Schwarz, H.; Schroeder,

D. Curr. Opin. Chem. Biol. 2002, 6, 556-567.

(14) Harris, N.; Cohen, S.; Filatov, M.; Ogliaro, F.; Shaik, S. Angew.

Chem., Int. Ed. 2000, 39, 2003-2007.

(15) Boddupalli, S. S.; Pramanik, B. C.; Slaughter, C. A.; Estabrook,

R. W.; Peterson, J. A. Arch. Biochem. Biophys. 1992, 292, 20-28.

(16) Capdevila, J. H.; Wei, S.; Helvig, C.; Falck, J. R.; Belosludtsev,

Y.; Truan, G.; Graham-Lorence, S. E.; Peterson, J. A. J. Biol. Chem.

1996, 271, 22663-22671.

(17) Bower, S.; Perkins, J. B.; Yocum, R. R.; Howitt, C. L.; Rahaim,

P.; Pero, J. J. Bacteriol. 1996, 178, 4122-4130.

(22) Koo, L. S.; Immoos, C. E.; Cohen, M. S.; Farmer, P. J.; Ortiz de

Montellano, P. R. J. Am. Chem. Soc. 2002, 124, 5684-5691.

(23) McLean, M. A.; Maves, S. A.; Weiss, K. E.; Krepich, S.; Sligar,

S. G. Biochem. Biophys. Res. Commun. 1998, 252, 166-172.

(24) Cryle, M. J.; Stuthe, J. M. U.; Ortiz de Montellano, P. R.; De

Voss, J. J. Chem. Commun. 2004, 512-513.

(25) The probes are actually C₁₅or C₁₇compounds but are considered

as derivatives of C₁₄or C₁₆fatty acids as this represents the longest

carbon chain in the molecule.

2456 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

SCHEME 1. Synthesis of the Cyclopropyl Fatty

Acids 1a and 3a and the ω-2 Hydroxycyclopropyl

Fatty Acids 5a and 6a^a

SCHEME 2. Synthesis of the ω-1 Hydroxylated

Cyclopropyl Fatty Acid Ester 9^a

^aReagents and conditions: for R ) CH₃CH₂CH₂- series: (a)

n-BuLi, THF, HMPA -40 °C, then CH₃CH₂CH₂Br, 81%. (b) (i)

p-TsOH, MeOH; (ii) Z: H₂, Lindlar catalyst, hexanes, 86%; E: Li,

NH₃, t-BuOH, THF, -40 °C, 68%. (c) CH₂I₂, Et₂Zn, TFA, CH₂Cl₂,

0 °C, 97-100%. (d) CrO₃, H₂SO₄, Me₂CO, 0 °C, 70-91%. (e) (i)

(COCl)₂, DMSO, CH₂Cl₂, -78 °C, then TEA; (ii) Ph₃PCHCO₂Me,

CHCl₃, reflux; (iii) H₂, PtO, hexanes; (iv) LiOH, MeOH, 10-15%

(four steps). For R ) CH₃CH₂CH(OH)- series: (a) n-BuLi, THF,

-40 °C, then EtCHO, 99%. (b) Z: H₂, Lindlar catalyst, hexanes,

100%; E: LAH, THF, 0 °C, 99%. (c) CH₂I₂, Et₂Zn, TFA, CH₂Cl₂, 0

°C, 98-100%. (d) (i) p-TsOH, MeOH; (ii) CrO₃, H₂SO₄, acetone, 0

°C; (iii) NaBH₄, CeCl₃, MeOH, 0 °C, 69-83% (three steps). (e) (i)

(COCl)₂, DMSO, CH₂Cl₂, -78 °C, then TEA; (ii) Ph₃PCHCO₂Me,

CHCl₃, reflux; (iii) NaBH₄, CeCl₃, MeOH, 0 °C; (iv) H₂, PtO,

hexanes; (v) LiOH, MeOH, 17-19% (five steps).

^aReagents and conditions: (a) Ph₃P(CH₂)₃OH, n-BuLi, Et₂O,

-78 °C, 86%. (b) PCC, NaAc, CH₂Cl₂, 94%. (c) BrMgCH₃, Et₂O,

87%. (d) p-TsOH, MeOH, 91%. (e) CH₂I₂, Et₂Zn, TFA, CH₂Cl₂, 0

°C, 89%. (f) (i) CrO₃, H₂SO₄, Me₂CO, 0 °C; (ii) CH₂N₂, Et₂O; (iii)

NaBH₄, CeCl₃, MeOH, 0 °C, 90% (two steps).

chosen to install the hydroxyl moiety, is known to proceed

with a strong preference for production of the anti

diastereomer,²⁹we were also able to use the products 5

and 7 to establish the diastereoselectivity of the P450

mediated oxidation.

The other likely unrearranged products from P450

mediated oxidation of 1a and 3a correspond to ω-1

oxidation. The aldehyde 38, available via ozonolysis of

protected undec-11-en-1ol, provided the differentially

protected terminal diol 43 as a 1:1 mixture of E and Z

isomers after Wittig chain extension (Scheme 2). Methyl

Grignard addition to the corresponding aldehyde, cyclo-

propyl ring formation, and manipulation of the oxidation

states of the oxygenated carbons provided the hydroxy

ester 9 as an equal mixture of all four diastereomers. As

expected, none of the distereoselectivity seen in the

reduction of the R-cyclopropyl ketones precursors to 5a

and 7a was observed in this series.

Oxidative rearrangement of the disubstituted cyclo-

propanes 1a and 3a could yield either a primary (11a,

12a) or a secondary (13a, 14a) alcohol, depending on the

ring scission pathway followed (Figure 2). The products

from the C₁₄series were prepared as primary standards

as well as to provide an indication of GCMS fragmenta-

tion patterns for the C₁₆series.

The acid 13a is the most likely product of oxidative

rearrangement of 1a as the rate of ring opening of the

corresponding cyclopropyl carbinyl radical to afford a

secondary radical (Figure 2, path b) is faster than the

alternative fragmentation that leads to 11a (Figure 2,

path a). Synthesis of 13 was achieved via allylmagnesium

bromide addition to the aldehyde 38, which in turn was

derived from ozonolysis of THP-protected undec-10-en-

1-ol (Scheme 3). Subsequent ozonolysis of 39 and Wittig

chain extension gave the desired 15-carbon chain in 40

with the required unsaturation and oxygenation. Func-

enzyme.¹⁸While this work was ongoing, a report ap-

peared detailing the synthesis and unsuccessful use of

3a²⁶and other related compounds as probes of the

mechanism of a non-heme iron desaturase.²⁷In this case,

although enzymic products were observed, no rearrange-

ment products were detected.

The terminal acetylene 18 was considered a key

precursor in the expedient synthesis of both the disub-

stituted cyclopropanes 1a and 3a and their expected

oxidation products 5a and 7a, respectively (Scheme 1).

This was indeed the case as alkylation of 18 cleanly

provided the internal alkyne 19, which could be converted

selectively to the corresponding Z or E alkene 20 by

reduction with either hydrogen in the presence of Lind-

lar’s catalyst (>98% Z) or lithium in liquid ammonia

(>95% E). Simmons-Smith cyclopropanation²⁸of 20 and

oxidation of the alcohol terminus led directly to the C₁₄

probes 1a, while the C₁₆probes 3a required a two carbon

chain extension via a Wittig condensation reaction.

An analogous route was used to synthesize products

5a and 7a expected from P450 mediated oxidation of 1a

and 3a except that it was initiated by addition of 18 to

the carbonyl of propanal. Fortuitously, as the sodium

borohydride reduction of cyclopropyl ketones, the method

(26) If an acid and the corresponding methyl ester are discussed in

this paper, a compound number followed by an “a” indicates the acid,

and the compound number alone indicates the ester.

(27) Villorbina, G.; Roura, L.; Camps, F.; Joglar, J.; Fabrias, G. J.

Org. Chem. 2003, 68, 2820-2829.

(28) Zhiqiang, Y.; Lorenz, J. C.; Shi, Y. Tetrahedron Lett. 1998, 39,

8621-8624.

(29) Kazuta, Y.; Abe, H.; Yamamoto, T.; Matsuda, A.; Shuto, S. J.

Org. Chem. 2003, 68, 3511-3521.

J. Org. Chem, Vol. 70, No. 7, 2005 2457

Cryle et al.

FIGURE 2. Possible products from the P450 mediated hydroxylation of cyclopropyl fatty acids 1a and 3a.

SCHEME 3. Synthesis of the Unsaturated

Hydroxyester 13^a

SCHEME 4. Synthesis of the Unsaturated

Hydroxy Ester 11^a

^aReagents and conditions: (a) (i) LDA, THF, -78 °C, then

Br(CH₂)₉OTHP; (ii) CuCl₂; (iii) NaBH₄, MeOH, 0 °C, 20% (three

steps). (b) TBDPSCl, imidazole, CH₃CN, 65%; (c) (i) p-TsOH,

MeOH; (ii) CrO₃, H₂SO₄, Me₂CO, 0 °C; (iii) CH₂N₂, Et₂O, 80%

(three steps). (d) TBAF, THF, 100%.

^aReagents and conditions: (a) AllylMgBr, Et₂O, -40 °C, 87%

(b) (i) O₃, CH₂Cl₂, -78 °C then DMS; (ii) Ph₃P(CH₂)₂CH₃, n-BuLi,

Et₂O, -78 °C, 72% (two steps). (c) p-TsOH, MeOH, 90%. (d) (i)

CrO₃, H₂SO₄, Me₂CO, 0 °C; (ii) CH₂N₂, Et₂O, 88%. (e) NaBH₄,

MeOH, 0 °C, 75%.

SCHEME 5. Synthesis of the Cyclopropyl Fatty

Acids 2a and 4a^a

tional group manipulation of 40 then gave 13 as a

mixture of E/Z isomers.

The rearrangement product 11a would be formed from

ring opening of the cyclopropylcarbinyl radical derived

from 1a to afford a primary radical (Figure 2). Alkylation

of the R,â-unsaturated dimethyl hydrazone derived from

hex-2-enal with THP protected 9-bromononan-1-ol to

yield a â,γ-unsaturated alcohol 35 after reduction was

the key step in the synthesis of 11 (Scheme 4).^30,31

Manipulation of protecting groups and adjustment of the

oxidation state of 35 provided the desired ester 11.

The monosubstituted cyclopropanes 2a and 4a were

synthesized via initial alkylation of THP protected pro-

pargyl alcohol with a 12-carbon ω-cyclopropyl bromide

22 (available in two steps from commercial undec-10-en-

1-ol) (Scheme 5). Following relevant functional group

manipulation, this afforded 2a and then 4a using Wittig

chain-extension methodology.

Ozonolysis of cyclododecene employing Schreiber’s³²

methodology produced differentiated, terminally difunc-

tionalized C₁₂compounds that allowed access to both

unrearranged and rearranged oxidation products arising

from 2a and 4a (Figure 3). Thus, acetal aldehyde 30

^aReagents and conditions: (a) (i) 1-(tetrahydropyran-2′-yloxy)-

prop-2-yne, n-BuLi, THF, HMPA, -40 °C, then 22, 85%. (b) H₂,

Pd/C, hexanes, 99%. (c) p-TsOH, MeOH, 98%. (d) CrO₃, H₂SO₄,

Me₂CO, 0 °C, 89%. (e) (i) PCC, CH₂Cl₂; (ii) Ph₃PCHCO₂Me, CHCl₃,

reflux, 57% (two steps). (f) (i) H₂, Pd/C, hexanes; (ii) LiOH, MeOH,

64% (two steps).

(30) Mino, T.; Fukui, S.; Yamashita, M. J. Org. Chem. 1997, 62,

734-735.

(31) Yamashita, M.; Matsumiya, K.; Nakano, K. Bull. Chem. Soc.

Jpn. 1993, 66, 1759-1763.

smoothly undergoes vinyl Grignard addition and cyclo-

propanation as the key steps in the synthesis of 6a and

8a (Scheme 6). The product expected from oxidative

(32) Schreiber, S. L.; Claus, R. E.; Reagan, J. Tetrahedron Lett. 1982,

23, 3867-3870.

2458 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

FIGURE 3. Possible products from the P450 mediated hydroxylation of cyclopropyl fatty acids 2a and 4a.

SCHEME 6. Synthesis of the ω-2

SCHEME 8. Equilibration of Cyclopropyl Cation

in the Rearrangement of 5a

Hydroxycyclopropyl Fatty Acids 6a and 8a^a

(Scheme 8) and more slowly undergoes ring opening to a

homoallylic cation that can be quenched to yield the ob-

served unsaturated alcohols. The 1:1 ratio of these

homoallylic alcohols mandates a much faster rate of equil-

ibration than of ring opening for the cyclopropyl cations.

Such a rapid equilibration between degenerate cations has

previously been observed spectroscopically by Olah in

superacid solution.³³Importantly, if a cationic ring open-

ing is proposed as the origin for any homoallylic alcohol

13a observed in a P450 mediated oxidation of 1a, it would

be expected to be accompanied by an equal amount of the

isomeric 53a, as well as the rearranged cyclopropyl alcohol

54a. This would analogously hold for the oxidation of 3a.

Although it is possible that the enzyme active site could

impose significant restraints upon the molecular motion

of any intermediate and thus direct the product distribu-

tion, the lack of any cation derived product in both the

C₁₄and C₁₆equivalent series makes this an unlikely

explanation. In addition, active site constraints have

previously been discounted with other cyclopropyl con-

taining P450 substrates.³⁴Thus, 1a and 3a should not

only provide a means of detection of a radical or cationic

intermediate in a P450 mediated oxidation, but they

should also serve to distinguish between the two.

^aReagents and conditions: (a) BrMgCHdCH₂, Et₂O, 92%. (b)

CH₂I₂, Et₂Zn, TFA, CH₂Cl₂, 0 °C, 92%. (c) p-TsOH, Me₂CO, 97%.

(d) (i) CrO₃, H₂SO₄, Me₂CO, 0 °C; (ii) NaBH₄, CeCl₃, MeOH, 0 °C,

62%. (e) Ph₃PCHCO₂Me, CHCl₃, reflux, 79%. (f) (i) H₂, Pd/C,

hexane; (ii) LiOH, MeOH, 55% (two steps).

SCHEME 7. Synthesis of the Unsaturated

Hydroxyester 15^a

^aReagents and conditions: (a) Ph₃P(CH₂)₃OH, n-BuLi, Et₂O,

-78 °C, 60%.

rearrangement of 2a is 15a (Figure 3), and the corre-

sponding methyl ester standard 15 was afforded in one

step via a selective Wittig condensation with methyl 12-

oxododecanoate 48, also available via ozonolysis of cy-

clododecene³²(Scheme 7).

GCMS Analysis of Cyclopropyl Fatty Acid Esters.

GCMS analysis of the methyl esters of the initially

produced acids was used as the major method for

identification of the structures of the products from P450

mediated oxidation of 1a-4a. A number of fragmentation

patterns characteristic of the position of hydroxylation

along a saturated fatty acid ester chain have previously

been reported by us¹⁸and others^35-37(Scheme 9). Gener-

ally, major peaks are seen for scission R to the hydroxyl

Chemical Rearrangement of Cyclopropyl Cation.

The products of rearrangement of the cyclopropyl cation

derived from 1a under Lewis acidic conditions were

investigated. It was found that two isomeric homoallylic

alcohols and two isomeric cyclopropyl alcohols were

present in the reaction mixture, both in a 1:1 ratio.

Separation of these two pairs of compounds by chroma-

tography on silver impregnated silica gel after esterifi-

cation with diazomethane and extensive 2-D NMR ex-

periments identified the homoallylic alcohols as 13 and

53 and the cyclopropyl containing compounds as 1 and

54. This suggests that the initially formed cation rapidly

equilibrates between the two degenerate, stabilized forms

(33) Olah, G. A.; Reddy, V. P.; Prakash, G. K. S. Chem. Rev. 1992,

92, 69-95.

(34) Newcomb, M.; Le Tadic, M.-H.; Putt, D. A.; Hollenberg, P. F.

J. Am. Chem. Soc. 1995, 117, 3312-3313.

J. Org. Chem, Vol. 70, No. 7, 2005 2459

Cryle et al.

SCHEME 9. Major MS Fragmentation Pathways of Hydroxy Fatty Acid Methyl Esters

TABLE 1. Regioselectivity of Hydroxylation by P450_BM3of C₁₄and C₁₆Straight Chain Fatty Acids and Cyclopropyl

Fatty Acids 1a-4a

percent hydroxylation

probe

coupling⁴⁵(%)

>ω-3 (%)

ω-3 (%)

21 ( 2

ω-2 (%)

ω-1 (%)

tetradecanoate

Z-1a

70

73

0

32 ( 2

47 ( 3

66 ( 9

33 ( 2 (21:12)^a

1.0 ( 0.1 13a

70 ( 4 (1:1)^a

86 ( 4

E-1a

70

80

68

(ω-5) 11 ( 1

19 ( 1

22 ( 2

50 ( 8

0

2a

0

14 ( 1

33 ( 3

hexadecanoate

(ω-5) 0.5

(ω-4) 1.5

0

43 ( 3

Z-3a

75

48 ( 1 (2:1)^a

1.5 ( 0.1 14a

100 (syn)^a

89 ( 3

E-3a

80

55

0

4a

11 ( 1

^aSyn to anti ratio displayed in parentheses.

with resultant loss of the alkyl chain (pathway A, Scheme

9) and lactonization and subsequent R scission (pathway

B, Scheme 9). For hydroxy fatty acid esters where the

hydroxyl is in the ω or ω-1 position, hydrogen atom

transfer and loss of an aldehyde is observed (pathway

C, Scheme 9). It was important to clarify whether these

major fragmentation patterns were still observed in

hydroxy fatty acid esters bearing cyclopropyl (or alkene)

moieties near the alcohol group, which was permitted via

GCMS analysis of the suite of synthetic standards

synthesized previously. The fragmentations of Scheme

9 in the main provided explanations for the fragments

seen in these compounds, although the presence of the

cyclopropyl could effect partitioning between the A/B or

the C pathway in some cases and induce novel pathways

in others. Thus, a cyclopropyl R to the hydroxyl and away

from the carboxyl terminus of the chain induces frag-

mentation by pathway C (6 and 8 Figure 4). Functionality

â to the hydroxyl but still distal to the carboxyl moiety

has little effect, and paths A/B are observed (13 and 14

Figure 4). If the cyclopropyl ring is proximal to the

carboxyl terminus and R to the hydroxyl paths, A/B

operates although it is clear that alternative fragmenta-

tion pathways also begin to compete with these (5 and 7

Figure 4). If the functionality is proximal to the carboxyl

but â to the hydroxyl, then the major fragments observed

generally arise from a novel pathway, corresponding to

loss of methanol and a terminal aldehyde, presumably

via directed fragmentation of an intermediate lactone (9

and 10 Figure 4). These generalizations hold true not

only for the synthetic standards but for all of the

identified products of enzymic oxidation and chemical

rearrangement seen in this work (vide infra) (Figure 4).

Enzyme Catalyzed Oxidation of Cyclopropyl Fatty

Acids. All of the synthesized cyclopropyl fatty acids 1a-

4a were incubated with P450_BM3, P450_BioI, and CYP119

in the presence and absence of NADPH (or NADH), and

for P450_BioIand CYP119, appropriate electron-transfer

partners. P450_BM3oxidized all of the fatty acids, P450_BioI

oxidized all but E-3a, while CYP119 only gave detectable

products with Z-1a, 2a, and E-3a. The products were

identified by GCMS analysis, and where appropriate,

comparison with synthetic standards and the regiochem-

istry of oxidation is given in Tables 1-3. The levels of

turnover were such that mechanistic information could

be derived for only P450_BM3and P450_BioI, but some

information on the influence of the cyclopropyl moiety

on the regiochemistry of oxidation for all three enzymes

(35) Kawamura, K.; Gagosian, R. B. J. Chromatogr. 1988, 438, 309-

317.

(36) Kerwin, J. L.; Torvik, J. J. Anal. Biochem. 1996, 237, 56-64.

(37) Wilson, R.; Smith, R.; Wilson, P.; Shepherd, M. J.; Riemersma,

R. A. Anal. Biochem. 1997, 248, 76-85.

2460 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

FIGURE 4. Major fragmentation pathways and ions seen in mass spectra of a variety of hydroxy fatty acid esters.

TABLE 2. Regioselectivity of Hydroxylation by P450_BioIof C₁₄and C₁₆Straight Chain Fatty Acids and Cyclopropyl

Fatty Acids 1a-4a

percent hydroxylation

probe

tetradecanoate¹⁸

coupling⁴⁵(%)

>ω-3 (%)

ω-3 (%)

49 ( 1

ω-2 (%)

ω-1 (%)

6

8

0

30 ( 1

21 ( 1

41 ( 3

Z-1a

58 ( 1 (37:21)^a

1.6 ( 0.3 13a

77 ( 7 (2:1)^a

31 ( 4

E-1a

2a

7

6

2

0

23 ( 4

15 ( 2

9 ( 2

0

69 ( 11

24 ( 2

hexadecanoate¹⁸

(ω-5) 12 ( 2

29 ( 2

(ω-4) 20 ( 2

Z-3a

13

(ω-5) 73 ( 5 (4:1)^a50a

16 ( 2 (1:1)^a

2.2 ( 0.8 17a

E-3a

4a

0

<1

0

∼10

∼90

^aSyn to anti ratio displayed in parentheses.

TABLE 3. Regioselectivity of Hydroxylation by CYP119 of C₁₄and C₁₆Straight Chain Fatty Acids and Cyclopropyl

Fatty Acids Z-1a, 2a, and E-3a

percent hydroxylation

probe

>ω-3 (%)

ω-3 (%)

ω-2 (%)

ω-1 (%)

tetradecanoate

(ω-5) 13

17

4

0

(ω-4) 66

Z-1a

2a

0

∼20

100

∼80

0

percent hydroxylation

probe

ω-7 (%)

ω-6 (%)

ω-5 (%)

ω-4 (%)

hexadecanoate

E-3a

17

0

76

trace

7

0

is available. The results will be discussed under these

two broad categories.

the ω-5 position and yields 50a, whereas for Z-1a reaction

occurs at the ω-2 carbon (5a) (Table 2). With P450_BM3

,

Mechanism of P450sa Radical But Not a Cation

Intermediate. The products from the P450 catalyzed

oxidation of compounds 1a-4a were carefully examined

for both the products of direct hydroxylation R to the

cyclopropyl ring and the rearrangement products arising

from either radical or cationic intermediates involved in

this oxidation. Because of the relative rates of cyclopropyl

ring opening,^6,7the most likely compounds to exhibit

rearrangement products were Z-1a and Z-3a predicted

to ring open 4 times faster than 2a and 4a and >3 times

faster than E-1a and E-3a. Previously, we reported that

Z-1a and Z-3a, when incubated with P450_BM3, did indeed

produce both the R-hydroxylated products 5a and 7a as

well as the rearranged products 13a and 14a, respec-

tively.²⁴None of the other products (53a or 54a) expected

from rearrangement of a cationic intermediate were

observed, and the ratio of the rearranged to nonrear-

ranged products allowed an upper limited of 2.6 × 10¹⁰

s^-1to be calculated for the rate of oxygen rebound.²⁴We

now report that the incubation of Z-1a and Z-3a with

P450_BioIagain results in high levels of oxidation R to the

cyclopropyl ring, although in the case of Z-3a this is at

oxidation only occurred at the ω-1 position in both Z-1a

and Z-3a (Table 1). Along with the products of direct

hydroxylation of Z-1a and Z-3a, the homoallylic alcohols

expected from rearrangement of an intermediate radical

were also observed. No isomeric cyclopropyl or homoal-

lylic alcohols expected from rearrangement of cationic

intermediates were detected in any of the enzymic

reactions. Quantitation (Table 2) of the R-cyclopropyl

alcohol and the corresponding rearranged homoallylic

alcohol (Z-1/13 and Z-3/50) and utilization of a ring

opening rate of 8.0 × 10⁸s^-1for Z-disubstituted cyclo-

propanes^6,7allowed calculation of the rebound rate for

the Fe(IV)-OH and the carbon radical pair in each case.

The rates obtained were in excellent internal agreement

(2.9 × 10¹⁰s^-1for Z-1a and 2.7 × 10¹⁰s^-1for Z-3a) with

each other and with the results obtained for P450_BM3

oxidation of the same probes (2.6 × 10¹⁰s^-1).

The two state reactivity theory of P450 mechanism

suggests that the nature and structure of the substrate

may influence partitioning between the nonconcerted and

effectively concerted pathways. This has been used to

explain the very different rebound rates calculated from

J. Org. Chem, Vol. 70, No. 7, 2005 2461

Cryle et al.

data obtained with different cyclopropyl containing

probes.³It was thus important to determine whether the

structural differences between Z-1a/Z-3a and E-1a/E-

3a and the monosubstituted cyclopropanes 2a and 4a,

which lead to significant differences in the rate of ring

opening, led to different apparent rebound rates. No

rearranged homoallylic alcohol was detected in the P450

catalyzed oxidation of 2a or 4a, but this was to be

expected based upon the rebound rate calculated previ-

ously, the expected rate of ring opening, and the sensitiv-

ity of detection. However, in the oxidation of E-1a and

E-3a by P450_BM3, the rearranged homoallylic alcohols 13

and 14 were detectable by analysis of single ion chro-

matograms following after esterification with diaz-

omethane, corresponding to their major fragment ions.

This was necessary as 13 and 14 coeluted under analysis

conditions with the major ω-2 oxidation products 5 and

7, respectively, which unfortunately also precluded ac-

curate quantitation. A large scale oxidation of E-1a (0.02

mmol) by P450_BM3was conducted, the products of turn-

(E-1a, E-3a, 2a, and 4a) exhibited changes in regiochem-

istry attributable to weakening of the pseudo-allylic C-H

bonds R to the cyclopropyl ring.⁴⁰Such electronic effects

in determining the regiochemistry of P450 oxidation have

been reported previously, with an order of preference for

oxidation site in the absence of binding effects paralleling

bond strength (i.e., CH > CH₂> CH₃).^40-43In fact, in the

case of E-3, only the product of hydroxylation R to the

cyclopropyl is observed, an unprecedented regioselectivity

for fatty acid hydroxylation by this enzyme. The signifi-

cant amount of ω-5 oxidation observed with the relatively

short E-1a is similarly unusual in fatty acid hydroxy-

lation by wild-type P450_BM3. Epoxidation has been found

a similar distance from the methyl terminus of aracha-

donic acid,¹⁶and very low levels of ω-5 hydroxylation have

previously been reported for oxidation of the longer

palmitate.^39,44Collectively, the results demonstrate that

a somewhat surprising degree of flexibility must exist for

the binding of substrates by P450_BM3. The fatty acids

Z-1a and Z-3a would be conformationally distinct from

the other fatty acids used in this study as well as straight

chain ones because of the Z ring geometry, which would

be expected to significantly reduce the binding alterna-

tives of the substrates to the enzyme. The interplay of

electronic and binding effects in these cases results in a

significant amount of oxidation both R to the cyclopropyl

in the electronically favored position and at the sterically

more accessible â carbon.

P450_BioIis known to be much less regioselective and

enantioselective than P450_BM3in its oxidation of straight

chain fatty acids.¹⁸Moderate enantioselectivity is seen

across the ω-1 to ω-3 positions in tetradecanoic acid,

while similar enantioselectivity is seen from the ω-1 to

ω-5 position in hexadecanoic acid.¹⁸The lack of regiose-

lectivity of P450_BioIis even more striking in that the C7-

C8 bond cleavage via a cascade of oxidative steps in

tetradecanoic acid is also seen and is believed to be

related to its physiological function.²⁰Additionally, al-

though fatty acids bind well, their natural substrate is

believed to be a fatty acid thioester of an acyl carrier

protein.¹⁹Not surprisingly, therefore, the results of

P450_BioIcatalyzed oxidation of the probes were varied.

The disubstituted cyclopropyl fatty acids 1a and 3a in

general showed increased oxidation at the pseudoallylic

positions R to the three membered ring. The C₁₄equiva-

lent acids 1a and 2a were better substrates than analo-

gous straight chain tetradecanoic acid and the C₁₆

equivalent acids 3a and 4a, and in fact, E-3a was not

oxidized at all by P450_BioI. In the shorter probe 1a,

oxidation was predominantly at the ω-2 position, while

in the longer Z-3a it was at the ω-5 position proximal to

the carboxyl terminus. The monosubstituted cyclopropyl

fatty acids 2a and 4a were oxidized at the presumably

more accessible ω-3 position rather than the more reac-

tive ω-2 position R to the cyclopropyl ring.

1

over were analyzed using 500 MHz H NMR, and the

peaks corresponding to the vinylic hydrogens of 13 and

the methine hydrogens R to both the hydroxyl and the

cyclopropyl of 5 were quantified by integration. These

imprecise measurements suggested a rebound rate of 2.2

× 10¹⁰s^-1(see Supporting Information).

The close correspondence of the rebound rates calcu-

lated for Z-1a and Z-3a oxidation with both P450_BM3and

P450_BioIand the qualitatively similar results obtained

with E-1a, which undergoes ring opening considerably

more slowly, is striking. Rebound rates from the same

probe in different enzymes,^5,10or from very similar probes

with the same enzyme,³⁸have often been significantly

different. Two state reactivity theory explains the sig-

nificant differences observed by suggesting that the

differences in enzyme or substrate structure determine

the flux through nonconcerted and effectively concerted

pathways and thus alter the observed rebound rate. The

results reported in this work suggest that for a matched

enzyme substrate pairsboth P450_BM3and P450_BioInatu-

rally utilize fatty acids, and the probes used here closely

resemble the natural substratessthe differences in path-

way utilization are small.

Effect of Cyclopropyl Moiety on the Regiochem-

istry of Hydroxylation. The products from P450_BM3

,

P450_BioI, and CYP119 catalyzed oxidation of 1a-4a

allowed not only determination of the presence and

lifetime of a radical intermediate but also demonstrated

the effect a cyclopropyl moiety in the fatty acid chain has

upon the regiochemistry of oxidation. P450_BM3binds

straight chain fatty acids, oxidizes them in a tightly

coupled fashion (NADPH consumption: product forma-

tion ≈1), and is known to catalyze their oxidation (at the

ω-1 to ω-3 positions) with moderate regioselectivity and

high enantioselectivity.^15,16,39All of the probes used in this

study were oxidized in a highly coupled fashion by

P450_BM3. The cyclopropyl fatty acids E-1a, E-3a, 2a, and

4a would be expected to have extended, albeit somewhat

rigidified, conformations analogous to those seen in

straight chain fatty acids. Oxidation of these fatty acids

(40) de Visser, S. P.; Kumar, D.; Cohen, S.; Shacham, R.; Shaik, S.

J. Am. Chem. Soc. 2004, 126, 8362-8363.

(41) Korzekwa, K. R.; Jones, J. P.; Gillette, J. R. J. Am. Chem. Soc.

1990, 112, 7042-7046.

(42) White, R. E.; McCarthy, M. B.; Egeberg, K. D.; Sligar, S. G.

Arch. Biochem. Biophys. 1984, 228, 493-502.

(38) He, X.; Ortiz de Montellano, P. R. J. Biol. Chem. 2004, 279,

39479-39484.

(39) Truan, G.; Komandla, M. R.; Falck, J. R.; Peterson, J. A. Arch.

Biochem. Biophys. 1999, 366, 192-198.

(43) Frommer, U.; Ullrich, V.; Staudinger, H. Hoppe-Seyler’s Z.

Physiol. Chem. 1970, 351, 903-912.

(44) Murataliev, M. B.; Trinh, L. N.; Moser, L. V.; Bates, R. B.;

Feyereisen, R.; Walker, F. A. Biochemistry 2004, 43, 1771-1780.

2462 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

19

5

Enzyme Expression and Purification. P450_BioI

,

P450_BM3,

CYP119 has previously been reported to bind fatty

acids of varying chain length and to oxidize lauric acid

with moderate regioselectivity (ω/ω-1/ω-2/ω-3 7:70:22:1).²²

The data obtained in this work for the oxidation of tetra-

decanoate and hexadecanoate (Table 3) again show only

moderate regioselectivity. Interestingly, the major site

of hydroxylation in these three straight chain fatty acids

appears to be eight (C₁₄and C₁₆) or nine (C₁₂) methylenes

removed from the carboxyl terminus, suggesting a role

for this moiety in determining substrate binding and hy-

droxylation site selectivity. The CYP119 catalyzed oxida-

tion of 1a-4a was investigated, and Z-1a, 2a, and E-3a

were oxidized, although extremely poorly. In the oxidation

of Z-1a, only the product from ω-2 oxidation was detected,

while 2a gave 80% ω-2 oxidation with the remainder of

the hydroxylation occurring at the ω-3 carbon. The

observed changes in the regiochemistry of oxidation again

probably reflect the weakening of the pseudoallylic C-H

bonds R to the cyclopropyl. E-3a gave a trace of a product,

tentatively identified by MS fragmentation as bearing the

hydroxyl at the ω-6 position. The levels of coupling were

so low, however, that accurate quantitation was not

possible, and the presence of other minor products cannot

be excluded at this stage. The regiochemistry of oxidation

of Z-1a is promising with respect to its use as a radical

clock for this enzyme. The improvements in catalytic

efficiency expected for the thermophilic CYP119 at

elevated temperatures along with the recently reported

thermally stable redox partners may allow Z-1a to be

used as a mechanistic probe for this enzyme.^21,22,46

Conclusion. The cyclopropyl containing fatty acids

1a-4a reported here will hopefully prove useful as

general mechanistic probes for the P450s and other

enzymes. Their ability to distinguish between cationic

and radical intermediates should prove particularly

valuable in this regard. Here, they have demonstrated

that a radical but not a cationic intermediate is formed

and CYP119⁴⁷were expressed and purified as previously

reported.

Enzymic Turnover. Incubation of cyclopropyl probes 1a-

4a with P450_BioI¹⁸(15 µM enzyme/1000 µM substrate/1250 µM

24

NADPH) and P450_BM3(2 µM enzyme/500 µM substrate/600

µM NADPH) was performed at 37 °C for 30 min as reported

previously. Coupling is reported as the ratio of product

formation to NADPH consumption (55-80% P450_BM3; 0-13%

P450_BioI), and high percentage coupling corresponds to only

small quantities of residual substrate remaining. Enzymatic

turnover with CYP119: turnovers of 1a-4a were performed

in triplicate with two blanks using the following protocol:

CYP119 (2 µM), fatty acid (500 µM), putidaredoxin reductase

(2 µM), putidaredoxin (100 µM), catalase (1 µM), and super-

oxide dismutase (10 units) were combined in a Wheaton

reaction vial. Aliquots of NADH (10 mM) were added to the

solution every 2 h, while the turnover was incubated at 37 °C

for at total of 6 h. Phenylacetic acid (final concentration 50

µM) was then added as an internal standard. The reaction was

then acidified with HCl (1 M) extracted with diethyl ether (2

× 1 mL) and esterified with diazomethane. The turnover

mixtures were concentrated before being analyzed by GC/MS.

Blank turnovers were performed under identical conditions

with the exception of either NADH or CYP119.

Analysis of Turnovers by GC/MS. See Supporting Infor-

mation.

General Reaction Procedures. Procedure A: Addition

of Deprotonated Terminal Alkyne. To a solution of termi-

nal alkyne in THF (0.10 M) being stirred at -40 °C under a

nitrogen atmosphere was added dropwise n-butyllithium (1.4

mol equiv). Following deprotonation of the alkyne after being

stirred for 2 h, either HMPA (4% v/v) was added followed after

15 min by a solution of alkyl bromide in THF (3 mol equiv,

0.5 M) or a solution of aldehyde in THF (3 mol equiv, 0.5 M)

was added directly without HMPA. The solution was warmed

to room temperature and stirred for 12 h after which cold

saturated, aqueous NH₄Cl (80 mL) was added to quench the

reaction. The THF was removed in vacuo, and the aqueous

layer was extracted with diethyl ether (3 × 50 mL). The

organic layers were washed with 3 M aqueous LiCl (3 × 50

mL) and brine (50 mL) and dried over MgSO₄, and the solvent

was removed in vacuo to afford the crude product.

during their oxidation catalyzed by P450_BM3and P450_BioI

.

The rebound rate calculated from these data for the

recombination of the intermediate carbon radical and the

iron tethered oxygen was remarkably constant across

both enzymes, probe structure, and position of oxidation.

This suggests a homogeneity of mechanism across these

enzymes with fatty acid substrates and perhaps in

general across P450s with their natural substrates.

Studies of 1a-4a with P450s that are not normally

involved in fatty acid metabolism and of analogues of

them that are not usually good substrates for P450_BM3

and P450_BioImay indicate how important the enzyme/

substrate pair is to the observed mechanism. Our pre-

liminary results with CYP119 also indicate that Z-1a

may be of particular utility, if the level of oxidation can

be increased sufficiently to allow investigation of any

differential effect of temperature on the spin states of the

iron oxo proposed to be important by two-state reactivity.

These investigations are currently ongoing.

Procedure B. Acidic Cleavage of THP-Ether. To a

solution of THP-protected alcohol in methanol (∼0.1 M) was

added p-TsOH (catalytic), and the reaction stirred for 2 h

before being quenched by addition of solid NaHCO₃(excess).

The solvent was removed in vacuo, and the residue was

partitioned between water (30 mL)/diethyl ether (30 mL).

Following separation of the layers, the aqueous layer was

extracted with diethyl ether (3 × 30 mL), and the combined

organic layers were washed with brine (30 mL) and dried over

MgSO₄, and the solvent was removed in vacuo to afford the

desired alcohol.

Procedure C. Hydrogenation of Double/Triple Bonds.

To a solution of alkene/alkyne in hexanes (∼0.3 M) and

relevant hydrogenation catalyst (catalytic) was added the

following: for reduction to single bonds, the catalyst used was

either 5% Pd/C or PtO, while for reduction of alkynes to

Z-alkenes, Lindlar’s catalyst was used. The solution was

introduced to a 1 atm hydrogen atmosphere and left to stir

for 2 h at room temperature. The solution was filtered to

remove the catalyst before being concentrated in vacuo to

afford the desired alkane/alkene product.

Procedure D. Cyclopropanation of Alkene. To a solu-

tion of diiodomethane (6 mol equiv) in CH₂Cl₂(∼1 M) being

stirred at 0 °C under a nitrogen atmosphere was added diethyl

zinc (3 mol equiv), and the reaction was stirred for 10 min. A

Experimental Procedures

General Methods and Instrumentation. See Supporting

Information.

(45) Coupling is the ratio of product formation to NAD(P)H con-

sumption.

(46) Puchkaev, A. V.; Ortiz de Montellano, P. R. Arch. Biochem.

Biophys. 2005, 434, 169-177.

(47) Koo, L. S.; Tschirret-Guth, R. A.; Straub, W. E.; Moenne-Loccoz,

P.; Loehr, T. M.; Ortiz de Montellano, P. R. J. Biol. Chem. 2000, 275,

14112-14123.

J. Org. Chem, Vol. 70, No. 7, 2005 2463

Cryle et al.

solution of alkene in CH₂Cl₂(∼0.5 M, 1 mol equiv) was added,

and the reaction was stirred for 10 min before addition of a

solution of TFA in CH₂Cl₂(∼0.5 M, 1 mol equiv). The reaction

was allowed to stir at 0 °C for 1 h before being quenched by

addition of cold saturated, aqueous NH₄Cl (20 mL). Following

separation of the layers, the aqueous phase was extracted with

CH₂Cl₂(3 × 30 mL), and the combined organic layers were

washed with brine (50 mL) before being dried over MgSO₄.

Removal of the solvent in vacuo afforded the crude product.

Procedure E. Jones Oxidation. To a solution of alcohol

in acetone (∼ 0.1 M) being stirred at 0 °C was added Jones

reagent (8 N) until the solution remained orange. The reaction

was left to stir for 10 min before water (40 mL) was added,

and the aqueous layer was extracted with hexanes (4 × 30

mL). The combined organic layers were washed with brine (50

mL), dried over MgSO₄, and concentrated in vacuo to afford

the crude product.

Procedure F. Stabilized Wittig Reaction. To a solution

of aldehyde in chloroform (∼0.1 M) being stirred at room

temperature was added methoxycarbonylmethylene(triphenyl)-

phosphorane (2 mol equiv). The mixture was brought to reflux

for 2 h before being cooled to room temperature. Following

removal of the solvent in vacuo, the residue was passed

through a silica gel plug to afford the crude product.

Procedure G. Base-Catalyzed Ester Cleavage. The ester

was dissolved in methanol (∼0.1 M), and aqueous 1 M LiOH

(50% v/v) added dropwise while being stirred at room temper-

ature. The reaction was left to stir for 12 h before being cooled

to 0 °C and acidified to pH 1 with aqueous 1 M HCl. The

aqueous phase was extracted with ethyl acetate (4 × 20 mL),

and the combined organic layers were washed with brine (20

mL). After being dried over MgSO₄, the solvent was removed

in vacuo to afford crude product.

Procedure L. Nonstabilized Wittig Reaction. To a

solution of Wittig salt in diethyl ether (1.5 mol equiv, 0.1 M)

being stirred at room temperature under nitrogen was added

n-butyllithium (1.5 mol equiv). After 30 min, the reaction was

cooled to -78 °C before a solution of aldehyde was added in

diethyl ether (1.0 mol equiv, 0.25 M). After 2 h at -78 °C, the

reaction was warmed to room temperature and quenched with

ethanol (5 mL). Saturated, aqueous NH₄Cl (50 mL) and diethyl

ether (50 mL) were added, and the layers were separated. The

organic layer was extracted with diethyl ether (2 × 40 mL),

washed with brine (40 mL), and dried over Na₂SO₄, and the

solvent was removed in vacuo to afford the desired crude

product.

1-(Tetrahydropyran-2′-yloxy)-tetradec-10-yne (19). Pro-

cedure A, 81% yield from 1-(tetrahydropyran-2′-yloxy)-undec-

10-yne 18.^{48 1}H NMR (200 MHz, CDCl₃) δ 0.92 (3H, t, J₁) 7.3

Hz), 1.10-1.80 (22H, m), 2.09 (4H, m), 3.20-3.90 (4H, 2m),

4.51 (1H, t, J₁) 2.0 Hz). ¹³C NMR (50 MHz, CDCl₃) δ 13.4,

18.6, 19.6, 20.7, 22.5, 25.4, 26.1, 28.8, 29.0, 29.1, 29.36, 29.40,

29.7, 30.7, 62.2, 67.6, 79.9, 80.3, 98.7. GC/MS: 294 (M^+•, 0.1),

251 (1.1), 123 (2.3), 109 (4.2), 101 (22.5), 85 (100), 55 (33.2),

41 (57.4). Anal. Calcd. for C₁₉H₃₄O₂: C, 77.50; H, 11.64.

Found: C, 77.73; H, 11.28.

Tetradec-10-(Z)-en-1-ol (Z-20). Procedures B and C, 86%

yield from 19, >98% Z. ¹H NMR (400 MHz, CDCl₃) δ 0.85 (3H,

t, J₁) 7.4 Hz), 1.20-1.40 (13H, m), 1.45 (2H, quint, J₁) 4.7

Hz), 1.53 (2H, quint, J₁) 7.1 Hz), 2.09 (4H, m), 3.60 (2H, d of

d, J₁) 6.5 Hz, J₂) 5.4 Hz), 5.36 (2H, m). ¹³C NMR (100 MHz,

CDCl₃) δ 13.6, 22.7, 25.7, 28.8, 29.3, 29.40, 29.44, 29.5, 29.7,

32.8, 34.7, 63.0, 130.1, 130.5. GC/MS: 166 (1.5), 138 (1.8), 123

(2.4), 110 (4.8), 95 (12.8), 68 (45.1), 55 (91.1), 41 (100). Anal.

Calcd. for C₁₄H₂₈O: C, 79.18; H, 13.29. Found: C, 79.01; H,

13.24.

Tetradec-10-(E)-en-1-ol (E-20). The alcohol from 19 (0.550

g, 1.87 mmol) was prepared employing the general THP-

cleavage procedure B (0.337 g). To liquid ammonia (15 mL)

being stirred at -78 °C was added a solution of crude alcohol

(0.337 g) in t-butyl alcohol/THF (3:5, 8 mL). After 5 min,

lithium metal (0.080 g) was added, and the reaction was left

to stir for 1.5 h. The ammonia was then allowed to evaporate

after which saturated, aqueous NH₄Cl (30 mL) was added to

quench the reaction. The aqueous layer was extracted with

hexanes (4 × 30 mL) that were in turn washed with brine (3

× 30 mL). The organic layer was dried over MgSO₄and

concentrated in vacuo and purified by flash chromatography

(silica gel, 20% ethyl acetate in hexanes) to afford E-20 (0.269

Procedure H. PCC Oxidation of Alcohol. To a solution

of alcohol in CH₂Cl₂(0.1-0.01 M) being stirred at room

temperature under a nitrogen atmosphere was added PCC (2

mol equiv). After 1 h, the reaction mixture was reduced in

volume and passed through a silica gel plug to afford the crude

aldehyde/ketone.

Procedure I. NaBH₄Reduction of Ketone. To a solution

of ketone in methanol (∼0.1 M) was added either cerium

trichloride hexahydrate (1.2 mol equiv) and NaBH₄(5 mol

equiv) over 10 min for conjugated ketones or NaBH₄(5 mol

equiv) over 10 min for nonconjugated ketones. The solution

was allowed to stir for 2 h after which CH₂Cl₂(20 mL) was

added. The solution was then washed with aqueous, 5% oxalic

acid solution (20 mL), and the layers were separated. The

aqueous phase was extracted with CH₂Cl₂(2 × 20 mL), and

the combined organic layers were washed with saturated,

aqueous NaHCO₃solution (30 mL) and brine (30 mL), and the

organic layer was dried over MgSO₄. Concentration in vacuo

afforded the crude alcohol.

Procedure J. Grignard Addition to Carbonyl. To a

solution of aldehyde/ketone in THF (0.2 M) being stirred at

-40 °C under a nitrogen atmosphere was added dropwise a

solution of the Grignard reagent (2.0 mol equiv) over 30 min.

After being stirred overnight at room temperature, the solution

was quenched with saturated, aqueous NH₄Cl (80 mL), and

the layers were separated. The aqueous layer was extracted

with diethyl ether (2 × 60 mL), and the combined organic

layers were washed with brine (60 mL) and dried over MgSO₄,

and the solvent was removed in vacuo to afford the crude

product.

Procedure K. Ozonolysis of Double Bonds. Into a

solution of alkene in CH₂Cl₂(0.1 M) being stirred at -78 °C

was bubbled excess ozone gas. After the ozonide had formed,

the reaction was purged with O₂and nitrogen before dimethyl

sulfide (10% v/v) was added, and the reaction was warmed to

room temperature. After being stirred at room temperature

for 30 min, the solvent was removed in vacuo to afford the

desired crude aldehyde.

1

g, 1.27 mmol, 68% yield, >95% Z) as a clear, colorless oil. H

NMR (400 MHz, CDCl₃) δ 0.87 (3H, t, J₁) 7.4 Hz), 1.20-1.40

(13H, m), 1.47 (2H, quint, J₁) 5.4 Hz), 1.53 (2H, quint, J₁)

7.2 Hz), 1.97 (4H, quint, J₁) 6.5 Hz), 3.61 (2H, d of d, J₁)

6.6 Hz, J₂) 5.4 Hz), 5.33 (2H, m). ¹³C NMR (100 MHz, CDCl₃)

δ 13.8, 22.9, 25.7, 27.2, 29.25, 29.29, 29.39, 29.44, 29.5, 29.7,

32.8, 63.0, 129.6, 130.0. GC/MS: 166 (1.5), 138 (1.5), 123 (2.2),

110 (4.1), 95 (12.5), 68 (45.1), 55 (81.3), 41 (100). Anal. Calcd.

for C₁₄H₂₈O: C, 79.18; H, 13.29. Found: C, 78.78; H, 12.91.

9-(Z-2-Propylcyclopropyl)-nonan-1-ol (Z-21). Procedure

1

D, 97% yield from Z-20. H NMR (200 MHz, CDCl₃) δ -0.42

(1H, d of d of d, J₁) 8.5 Hz, J₂) 8.0 Hz, J₃) 4.0 Hz), 0.54

(3H, m), 0.84 (3H, t, J₁) 7.0 Hz), 1.00-1.35 (18H, m), 1.42

(2H, quint, J₁) 7.6 Hz), 2.87 (1H, br s), 3.51 (2H, t, J₁) 6.5

Hz). ¹³C NMR (50 MHz, CDCl₃) δ 10.7, 14.0, 15.3, 15.5, 23.1,

25.7, 28.6, 29.3, 29.4, 29.48, 29.53, 30.1, 30.7, 32.6, 62.5. GC/

MS: 208 (0.2), 183 (0.5), 123 (2.9), 109 (7.3), 95 (18.6), 82 (33.4),

41 (100). Anal. Calcd. for C₁₅H₃₀O: C, 79.58; H, 13.36. Found:

C, 79.29; H, 13.17.

9-(E-2-Propylcyclopropyl)-nonan-1-ol (E-21). Procedure

1

D, 100% yield from E-20. H NMR (200 MHz, CDCl₃) δ 0.03

(2H, t, J₁) 6.5 Hz), 0.32 (2H, m), 0.83 (3H, t, J₁) 7.2 Hz),

1.00-1.35 (18H, m), 1.44 (2H, quint, J₁) 6.5 Hz), 2.88 (1H,

(48) Hakogi, T.; Shigenari, T.; Katsumura, S.; Sano, T.; Kohno, T.;

Igarashi, Y. Bioorg. Med. Chem. Lett. 2003, 13, 661-664.

2464 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

br s), 3.52 (2H, t, J₁) 6.5 Hz). ¹³C NMR (50 MHz, CDCl₃) δ

11.6, 13.9, 18.4, 18.6, 22.6, 25.7, 29.38, 29.40, 29.42, 29.56,

29.59, 32.6, 34.2, 36.4, 62.5. GC/MS: 208 (0.2), 183 (0.7), 123

(2.8), 109 (7.0), 95 (17.1), 81 (30.9), 41 (100). Anal. Calcd. for

C₁₅H₃₀O: C, 79.58; H, 13.36. Found: C, 79.19; H, 13.48.

9-(Z-2-Propylcyclopropyl)-nonanoic Acid (Z-1a). Pro-

cedure E, 70% yield from Z-21, mp 40.0-42.0 °C. ¹H NMR

(400 MHz, CDCl₃) δ -0.35 (1H, d of d of d, J₁) 8.5 Hz, J₂)

8.0 Hz, J₃) 4.0 Hz), 0.55 (1H, d of d of d, J₁) 5.2 Hz, J₂)

5.2 Hz, J₃) 4.0 Hz), 0.64 (2H, m), 0.90 (3H, t, J₁) 7.6 Hz),

1.10-1.40 (16H, m), 1.61 (2H, quint, J₁) 6.4 Hz), 2.32 (2H, t,

J₁) 7.6 Hz), 10.0 (1H, br s). ¹³C NMR (100 MHz, CDCl₃) δ

10.9, 14.1, 15.5, 15.7, 23.3, 24.7, 28.7, 29.0, 29.2, 29.5, 29.6,

30.1, 30.9, 34.1, 180.6. Anal. Calcd. for C₁₅H₂₈O₂: C, 74.95; H,

11.74. Found: C, 75.16; H, 12.11.

Methyl 9-(Z-2-Propylcyclopropyl)-nonanoate (Z-1). Pre-

pared by derivatization of Z-1a with an ethereal solution of

diazomethane. GC/MS: 254 (M^+•, 0.2), 222 (2.5), 180 (2.9), 152

(2.6), 138 (3.8), 123 (5.8), 110 (7.6), 41 (100).

9-(E-2-Propylcyclopropyl)-nonanoic Acid (E-1a). Pro-

cedure E, 91% yield from E-21, mp 40.0-42.0 °C. ¹H NMR

(400 MHz, CDCl₃) δ 0.11 (2H, d of d, J₁) 6.5 Hz, J₂) 6.5

Hz), 0.34 (2H, d of d of d of d, J₁) 17.2 Hz, J₂) 8.9 Hz, J₃)

6.3 Hz, J₄) 4.5 Hz), 0.89 (3H, t, J₁) 7.6 Hz), 1.10-1.40 (16H,

m), 1.61 (2H, quint, J₁) 7.2 Hz), 2.32 (2H, t, J₁) 7.5 Hz),

10.0 (1H, br s). ¹³C NMR (100 MHz, CDCl₃) δ 11.7, 14.0, 18.5,

18.7, 22.8, 24.6, 29.0, 29.2, 29.4, 29.5, 29.6, 34.1, 34.3, 36.5,

180.7. Anal. Calcd. for C₁₅H₂₈O₂: C, 74.95; H, 11.74. Found:

C, 74.90; H, 12.10.

7.3 Hz), 1.10-1.40 (20H, m), 1.60 (2H, quint, J₁) 7.1 Hz),

2.32 (2H, t, J₁) 7.5 Hz), 8.10 (1H, br s). ¹³C NMR (100 MHz,

CDCl₃) δ 11.7, 14.0, 18.5, 18.7, 22.8, 24.7, 29.1, 29.2, 29.4, 29.5,

29.6, 29.66, 29.70, 34.0, 34.3, 36.6, 179.9. Anal. Calcd. for

C₁₇H₃₂O₂: C, 76.06; H, 12.01. Found: C, 75.77; H, 11.99.

Methyl 11-(E-2-Propylcyclopropyl)-undecanoate (E-3).

Prepared by derivatization of E-3a with an ethereal solution

of diazomethane. GC/MS: 282 (M^+•, 0.3), 251 (3.2), 250 (4.8),

208 (2.7), 166 (3.5), 110 (7.5), 97 (17.1), 55 (100).

12-Cyclopropyl-1-(tetrahydropyran-2′-yloxy)-dodec-2-

yne (23). Procedure A, 85% yield from 1-bromo-9-cyclopro-

pylnonane 22 (See Supporting Information). ¹H NMR (400

MHz, CDCl₃) δ -0.05 (2H, d of d of d, J₁) 4.8 Hz, J₂) 4.8

Hz, J₃) 1.5 Hz), 0.34 (2H, d of d of d, J₁) 8.4 Hz, J₂) 8.4

Hz, J₃) 1.9 Hz), 0.61 (1H, m), 1.10-1.50 (22H, m), 2.18 (2H,

t of t, J₁) 3.6 Hz, J₂) 1.1 Hz), 3.50 (1H, m), 3.81 (1H, m),

4.21 (2H, m), 4.78 (1H, t, J₁) 3.4 Hz). ¹³C NMR (100 MHz,

CDCl₃) δ 4.3 (2C), 10.9, 18.8, 19.1, 25.4, 28.6, 28.8, 29.1, 29.4,

29.48, 29.51, 29.6, 30.3, 34.7, 54.6, 61.9, 75.7, 86.7, 96.6. GC/

MS: 306 (M^+•, 0.1), 135 (2.6), 121 (4.2), 111 (7.8), 101 (19.7),

95 (23.3), 85 (100), 55 (52.8). Anal. Calcd. for C₂₀H₃₄O₂: C,

78.38; H, 11.18. Found: C, 78.59; H, 10.78.

12-Cyclopropyl-1-(tetrahydropyran-2′-yloxy)-dode-

cane (24). Procedure C, 99% yield from 23. ¹H NMR (400

MHz, CDCl₃) δ -0.04 (2H, d of d of d, J₁) 4.8 Hz, J₂) 4.8

Hz, J₃) 1.5 Hz), 0.34 (2H, d of d of d, J₁) 8.4 Hz, J₂) 8.4

Hz, J₃) 1.9 Hz), 0.61 (1H, m), 1.16 (2H, q, J₁) 7.2 Hz), 1.20-

1.40 (26H, m), 3.25-3.95 (4H, m), 4.56 (1H, t, J₁) 3.5 Hz).

¹³C NMR (100 MHz, CDCl₃) δ 4.3 (2C), 10.9, 18.9, 19.7, 25.5,

26.2, 26.7, 29.4, 29.49, 29.54, 29.6, 29.7, 29.8, 30.8, 34.8, 53.9,

61.9, 67.7, 98.8. GC/MS: 310 (M^+•, 0.3), 153 (1.0), 123 (1.2),

101 (10.4), 85 (100), 55 (36.9), 43 (32.4), 41 (50.5). Anal. Calcd.

for C₂₀H₃₈O₂: C, 77.36; H, 12.33. Found: C, 77.08; H, 12.07.

12-Cyclopropyldodecan-1-ol (25). Procedure B, 98% yield

Methyl 9-(E-2-Propylcyclopropyl)-nonanoate (E-1). Pre-

pared by derivatization of E-1a with an ethereal solution of

diazomethane. GC/MS: 254 (M^+•, 0.2), 222 (2.6), 180 (2.9), 152

(2.4), 138 (3.7), 123 (5.4), 110 (7.2), 41 (100).

11-(Z-2-Propylcyclopropyl)-undecanoic Acid (Z-3a). To

a solution of oxalyl chloride (0.105 mL, 1.11 mmol) in CH₂Cl₂

(5 mL) being stirred at -78 °C under a nitrogen atmosphere

was added dropwise DMSO (0.205 mL, 2.76 mmol). After being

stirred for 10 min, a solution of Z-21 (0.140 g, 0.614 mmol) in

CH₂Cl₂(1 mL) was added dropwise, and the reaction was left

to stir at -78 °C for 1 h. Triethylamine (1.33 mL, 9.82 mmol)

was added, and the reaction was allowed to warm to room

temperature. Brine (50 mL) and diethyl ether (20 mL) were

added, and following separation of the layers, the aqueous

phase was extracted with diethyl ether (2 × 20 mL). The

combined organic layers were dried over MgSO₄, and the

solvent was removed in vacuo to yield aldehyde (0.102 g) that

was used without further purification. Chain extension of the

aldehyde (0.102 g) was achieved via Wittig condensation

employing general procedure F, and the product was then

hydrogenated using the general procedure C using 5% Pd/C

catalyst. Base cleavage of the ester was accomplished using

the general procedure G. The residue was purified using flash

chromatography (silica gel, 20% ethyl acetate in hexanes, 1%

acetic acid) to afford Z-3a (0.030 g, 0.111 mmol, 10% yield over

1

from 24. H NMR (400 MHz, CDCl₃) δ -0.04 (2H, d of d of d,

J₁) 4.8 Hz, J₂) 4.8 Hz, J₃) 1.5 Hz), 0.34 (2H, d of d of d, J₁

) 8.4 Hz, J₂) 8.4 Hz, J₃) 1.9 Hz), 0.61 (1H, m), 1.16 (2H, q,

J₁) 7.2 Hz), 1.20-1.40 (21H, m), 3.60 (2H, t, J₁) 6.8 Hz).

¹³C NMR (50 MHz, CDCl₃) δ 4.3 (2C), 10.9, 18.9, 25.5, 26.2,

26.7, 29.4, 29.5, 29.6, 29.7, 29.8, 30.8, 34.8, 62.5. GC/MS: 180

(0.1), 152 (1.0), 124 (3.6), 123 (3.4), 109 (7.0), 95 (16.1), 55

(78.6), 41 (100). Anal. Calcd. for C₁₅H₃₀O: C, 79.58; H, 13.36.

Found: C, 79.70; H, 13.49.

12-Cyclopropyldodecanoic Acid (2a). Procedure E, 89%

yield from 25, mp 48.0-49.0 °C. ¹H NMR (400 MHz, CDCl₃) δ

-0.04 (2H, d of d of d, J₁) 4.8 Hz, J₂) 4.8 Hz, J₃) 1.5 Hz),

0.35 (2H, d of d of d, J₁) 8.4 Hz, J₂) 8.4 Hz, J₃) 1.9 Hz),

0.61 (1H, m), 1.15 (2H, q, J₁) 7.2 Hz), 1.20-1.55 (16H, m),

1.61 (2H, quint, J₁) 7.6 Hz), 2.33 (2H, t, J₁) 7.4 Hz), 10.10

(1H, br s). ¹³C NMR (100 MHz, CDCl₃) δ 4.3 (2C), 10.9, 24.7,

29.1, 29.2, 29.4, 29.5, 29.58, 29.63, 29.65, 29.67, 34.1, 37.8,

180.5. Anal. Calcd. for C₁₅H₂₈O₂: C, 74.95; H, 11.74. Found:

C, 75.00; H, 12.10.

Methyl 12-Cyclopropyldodecanoate (2). Prepared by

derivatization of 2a with an ethereal solution of diazomethane.

GC/MS: 224 (0.4), 180 (2.9), 152 (2.9), 138 (4.2), 123 (6.3), 110

(8.9), 98 (13.7), 41 (100).

Methyl 14-Cyclopropyltetradec-2-enoate (26). Proce-

dures H and F, 57% yield from 25. ¹H NMR (200 MHz, CDCl₃)

δ -0.04 (2H, d of d of d, J₁) 4.8 Hz, J₂) 4.8 Hz, J₃) 1.5 Hz),

0.36 (2H, d of d of d, J₁) 8.4 Hz, J₂) 8.4 Hz, J₃) 1.9 Hz),

0.62 (1H, m), 1.17 (2H, q, J₁) 7.2 Hz), 1.20-1.45 (16H, m),

1.61 (2H, quint, J₁) 7.6 Hz), 2.15 (2H, d of q, J₁) 1.5 Hz, J₂

) 7.0 Hz), 3.68 (3H, s), 5.78 (1H, d of t, J₁) 15.6 Hz, J₂) 1.5

Hz), 6.93 (1H, d of t, J₁) 15.8 Hz, J₂) 7.0 Hz). ¹³C NMR (50

MHz, CDCl₃) δ 4.3 (2C), 10.9, 24.7, 27.9, 29.1, 29.3, 29.4, 29.55,

29.58, 29.64, 29.7, 32.2, 37.2, 51.4, 120.7, 149.9, 167.2. Anal.

Calcd. for C₁₈H₃₂O₂: C, 77.09; H, 11.50. Found: C, 77.01; H,

11.23.

1

four steps) as a white solid, mp 45.0-47.0 °C). H NMR (400

MHz, CDCl₃) δ -0.34 (1H, d of d of d, J₁) 8.5 Hz, J₂) 8.0

Hz, J₃) 4.0 Hz), 0.54 (1H, d of d of d, J₁) 5.2 Hz, J₂) 5.2

Hz, J₃) 4.0 Hz), 0.64 (2H, m), 0.90 (3H, t, J₁) 7.0 Hz), 1.10-

1.40 (20H, m), 1.62 (2H, quint, J₁) 6.4 Hz), 2.32 (2H, t, J₁)

7.4 Hz), 10.9 (1H, br s). ¹³C NMR (100 MHz, CDCl₃) δ 10.9,

14.1, 15.5, 15.7, 23.3, 24.7, 28.7, 29.0, 29.2, 29.4, 29.5, 29.58,

29.60, 30.1, 30.9, 34.1, 180.6. Anal. Calcd. for C₁₇H₃₂O₂: C,

76.06; H, 12.01. Found: C, 76.09; H, 12.30.

Methyl 11-(Z-2-Propylcyclopropyl)-undecanoate (Z-3).

Prepared by derivatization of Z-3a with an ethereal solution

of diazomethane. GC/MS: 282 (M^+•, 0.1), 251 (2.5), 250 (4.1),

208 (2.2), 166 (3.0), 110 (6.9), 97 (16.5), 41 (100).

11-(E-2-Propylcyclopropyl)-undecanoic Acid (E-3a).

Procedure identical to Z-3a, 15% yield over four steps, mp

1

45.0-47.0 °C. H NMR (400 MHz, CDCl₃) δ 0.12 (2H, d of d,

14-Cyclopropyltetradecanoic Acid (4a). Procedures C

and G, 64% yield from 26, mp 54.0-55.0 °C. ¹H NMR (400

MHz, CDCl₃) δ -0.04 (2H, d of d of d, J₁) 4.8 Hz, J₂) 4.8

J₁) 6.5 Hz, J₂) 6.5 Hz), 0.35 (2H, d of d of d of d, J₁) 17.2

Hz, J₂) 8.9 Hz, J₃) 6.3 Hz, J₄) 4.5 Hz), 0.88 (3H, t, J₁)

J. Org. Chem, Vol. 70, No. 7, 2005 2465

Cryle et al.

Hz, J₃) 1.5 Hz), 0.36 (2H, d of d of d, J₁) 8.4 Hz, J₂) 8.4

Hz, J₃) 1.9 Hz), 0.62 (1H, m), 1.17 (2H, q, J₁) 7.2 Hz), 1.20-

1.45 (20H, m), 1.61 (2H, quint, J₁) 7.6 Hz), 2.32 (2H, t, J₁)

7.6 Hz), 8.80 (1H, br s). ¹³C NMR (100 MHz, CDCl₃) δ 4.3 (2C),

10.9, 24.7, 29.1, 29.2, 29.4, 29.55, 29.58, 29.63, 29.65, 29.66,

29.68, 29.71, 34.1, 37.8, 180.1. Anal. Calcd. for C₁₇H₃₂O₂: C,

76.06; H, 12.01. Found: C, 75.96; H, 11.63.

Methyl 14-Cyclopropyltetradecanoate (4). Prepared by

derivatization of 4a with an ethereal solution of diazomethane.

GC/MS: 282 (M^+•, 0.1), 250 (2.9), 208 (2.7), 166 (3.2), 152 (3.2),

111 (7.3), 98 (14.3), 41 (100).

1-(Tetrahydropyran-2′-yloxy)-tetradec-10-yn-12-ol (27).

Procedure A, 99% yield from 18.^{48 1}H NMR (400 MHz, CDCl₃)

δ 0.90 (3H, t, J₁) 6.9 Hz), 0.20-1.70 (21H, m), 1.82 (2H, m),

2.17 (2H, t of d, J₁) 7.1 Hz, J₂) 2.0 Hz), 3.30-3.90 (4H, m),

4.27 (1H, t of t, J₁) 6.4 Hz, J₂) 1.9 Hz), 4.55 (1H, t, J₁) 2.6

Hz). ¹³C NMR (100 MHz, CDCl₃) δ 9.4, 18.6, 19.7, 25.5, 26.2,

28.6, 29.0, 29.37, 29.39, 29.7, 29.8, 30.8, 31.2, 62.3, 63.9, 67.6,

81.1, 85.6, 98.8. GC/MS: 263 (0.1), 251 (0.3), 197 (5.6), 135

(1.8), 109 (3.6), 101 (23.3), 85 (100), 41 (47.3). Anal. Calcd. for

C₁₉H₃₄O₃: C, 73.50; H, 11.04. Found: C, 73.77; H, 11.38.

1-(Tetrahydropyran-2′-yloxy)-tetradec-10-(Z)-en-12-

ol (Z-28). Procedure C, 100% yield from 27, >98% Z. ¹H NMR

(400 MHz, CDCl₃) δ 0.86 (3H, t, J₁) 7.6 Hz), 1.20-1.70 (23H,

m), 2.03 (2H, quint, J₁) 6.9 Hz), 3.30-3.85 (4H, m), 4.30 (1H,

t of d, J₁) 9.6 Hz, J₂) 7.4 Hz), 4.53 (1H, t, J₁) 2.6 Hz), 5.31

(1H, t of d, J₁) 9.0 Hz, J₂) 7.6 Hz), 5.44 (1H, d of t, J₁)

11.0 Hz, J₂) 7.4 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 9.6, 19.6,

25.4, 26.2, 27.7, 29.2, 29.4, 29.5, 29.6, 29.7, 30.1, 30.3, 30.7,

62.2, 67.6, 69.0, 98.7, 132.3, 132.4. GC/MS: 201 (0.1), 199 (0.3),

135 (0.6), 123 (1.1), 109 (3.3), 101 (20.9), 85 (100), 41 (58.2).

Anal. Calcd. for C₁₉H₃₆O₃: C, 73.03; H, 11.61. Found: C, 72.95;

H, 11.73.

m), 4.54 (1H, t, J₁) 1.7 Hz). ¹³C NMR (100 MHz, CDCl₃) δ

9.7, 10.0, 17.0, 19.7, 25.1, 25.4, 26.2, 29.40, 29.43, 29.5, 29.7,

29.8, 30.0, 30.1, 30.8, 36.6, 62.3, 67.6, 77.7, 98.8. GC/MS: 241

(0.5), 213 (9.4), 109 (2.9), 101 (11.3), 95 (7.4), 85 (100), 57 (33.5),

41 (51.1). Anal. Calcd. for C₂₀H₃₈O₃: C, 73.57; H, 11.73.

Found: C, 73.40; H, 11.94.

anti-9-(2-(1-Hydroxypropyl)-Z-cyclopropyl)-nonano-

ic Acid (Z-5a). Procedures B, E, and I, 83% yield from Z-29,

1

mp 64.0-65.0 °C. H NMR (400 MHz, CDCl₃) δ -0.10 (1H, d

of d, J₁) 5.2 Hz, J₂) 5.2 Hz), 0.68 (1H, d of d of d, J₁) 8.4

Hz, J₂) 8.4 Hz, J₃) 4.5 Hz), 0.77 (1H, d of d of d of d, J₁)

11.6 Hz, J₂) 5.5 Hz, J₃) 5.0 Hz, J₄) 3.3 Hz), 0.86 (1H, d of

d of d, J₁) 9.1 Hz, J₂) 4.5 Hz, J₃) 1.0 Hz), 0.95 (3H, t, J₁

) 7.5 Hz), 1.20-1.40 (13H, m), 1.61 (4H, m), 2.30 (2H, t, J₁)

7.6 Hz), 3.11 (1H, d of t, J₁) 8.0 Hz, J₂) 6.6 Hz), 5.56 (1H,

br s). ¹³C NMR (100 MHz, CDCl₃) δ 9.7, 10.1, 22.4, 24.7, 28.6,

29.0, 29.2, 29.3, 29.36, 29.41, 29.5, 30.1, 34.1, 74.5, 179.4. Anal.

Calcd. for C₁₅H₂₈O₃: C, 70.27; H, 11.01. Found: C, 70.01; H,

10.77.

Methyl anti-9-(2-(1-Hydroxypropyl)-Z-cyclopropyl)-

nonanoate (Z-5). Prepared by derivatization of Z-5a with an

ethereal solution of diazomethane. GC/ MS: 252 (0.4), 241

(4.5), 209 (8.1), 173 (9.5), 169 (24.0), 149 (11.1), 81 (48.9), 41

(100).

anti-9-(2-(1-Hydroxypropyl)-E-cyclopropyl)-nonano-

ic Acid (E-5a). Procedures B, E, and I, 69% yield from E-29,

mp 64.0-65.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 0.30 (2H, d of

d of t, J₁) 25.3 Hz, J₂) 7.1 Hz, J₃) 5.8 Hz), 0.60 (2H, m),

0.93 (3H, t, J₁) 7.5 Hz), 1.19 (2H, q of d, J₁) 6.8 Hz, J₂) 2.6

Hz), 1.22-1.40 (11H, m), 1.57 (2H, quint, J₁) 7.2 Hz), 1.60

(2H, quint of d, J₁) 5.7 Hz, J₂) 2.0 Hz), 2.30 (2H, t, J₁) 7.5

Hz), 2.82 (1H, q of d, J₁) 5.6 Hz, J₂) 1.2 Hz), 5.88 (1H, br s).

¹³C NMR (100 MHz, CDCl₃) δ 10.0, 16.5, 17.1, 24.7, 29.1, 29.2,

29.3, 29.4, 29.6, 29.8, 30.0, 33.7, 34.1, 77.9, 179.4. Anal. Calcd.

for C₁₅H₂₈O₃: C, 70.27; H, 11.01. Found: C, 70.22; H, 11.36.

Methyl anti-9-(2-(1-Hydroxypropyl)-E-cyclopropyl)-

nonanoate (E-5a). Prepared by derivatization of E-5a with

an ethereal solution of diazomethane. GC/MS: 269 (0.4), 252

(0.3), 241 (10.4), 209 (16.4), 173 (10.8), 169 (11.7), 149 (9.0),

41 (100).

anti-11-(2-(1-Hydroxypropyl)-Z-cyclopropyl)-undecano-

ic Acid (Z-7a). Procedures B, H, F, C, I, and G, 19% yield

over six steps from Z-29, mp 68.0-69.0 °C. ¹H NMR (200 MHz,

CDCl₃) δ -0.09 (1H, d of d, J₁) 5.2 Hz, J₂) 5.2 Hz), 0.60-

0.90 (3H, m), 0.96 (3H, t, J₁) 7.4 Hz), 1.20-1.40 (16H, m),

1.60 (4H, m), 2.07 (1H, br s), 2.32 (2H, t, J₁) 7.4 Hz), 3.13

(1H, d of t, J₁) 9.0 Hz, J₂) 6.6 Hz), 5.95 (1H, br s). ¹³C NMR

(50 MHz, CDCl₃) δ 9.7, 10.1, 15.5, 22.4, 24.6, 28.6, 28.9, 29.1,

29.3, 29.4, 29.46, 29.54, 30.1, 30.6, 34.0, 74.5, 179.6. Anal.

Calcd. for C₁₇H₃₂O₃: C, 71.79; H, 11.34. Found: C, 72.07; H,

11.11.

Methyl anti-11-(2-(1-Hydroxypropyl)-Z-cyclopropyl)-

undecanoate (Z-7). Prepared by derivatization of Z-7a with

an ethereal solution of diazomethane. GC/MS: 280 (0.4), 269

(4.0), 237 (9.2), 201 (3.9), 197 (8.3), 177 (5.2), 121 (9.0), 41 (100).

anti-11-(2-(1-Hydroxypropyl)-E-cyclopropyl)-undecano-

ic Acid (E-7a). Procedures B, H, F, C, I, and G, 17% yield

over six steps from E-29, mp 68.0-69.0 °C. ¹H NMR (200 MHz,

CDCl₃) δ 0.32 (2H, d of d of t, J₁) 25.3 Hz, J₂) 7.1 Hz, J₃)

5.8 Hz), 0.59 (2H, m), 0.94 (3H, t, J₁) 7.5 Hz), 1.10-1.40 (17H,

m), 1.59 (4H, m), 2.31 (2H, t, J₁) 7.3 Hz), 2.82 (1H, q of d, J₁

) 6.6 Hz, J₂) 1.2 Hz), 5.22 (1H, br s). ¹³C NMR (50 MHz,

CDCl₃) δ 10.0, 16.6, 24.6, 25.0, 29.0, 29.1, 29.25, 29.33, 29.4,

29.5, 29.6, 29.8, 30.0, 33.7, 33.9, 77.9, 179.3. Anal. Calcd. for

C₁₇H₃₂O₃: C, 71.79; H, 11.34. Found: C, 71.69; H, 11.73.

Methyl anti-11-(2-(1-Hydroxypropyl)-E-cyclopropyl)-

undecanoate (E-7). Prepared by derivatization of E-7a with

an ethereal solution of diazomethane. GC/MS: 280 (0.3), 269

(7.8), 237 (16.8), 201 (4.2), 177 (3.9), 135 (7.8), 121 (8.6), 41

(100).

1-(Tetrahydropyran-2′-yloxy)-tetradec-10-(E)-en-12-

ol (E-28). To a solution of lithium aluminum hydride (0.090

g, 24.6 mmol) in THF (60 mL) being stirred at 0 °C under a

nitrogen atmosphere was added dropwise a solution of 27

(0.765 g, 2.46 mmol) in THF (20 mL). The solution was brought

to reflux for 1 h before the solution was cooled to room

temperature, and sodium sulfate decahydrate (excess) was

added over 2 h to quench the reaction. The solution was then

filtered, and the salts were washed with ethyl acetate (5 × 30

mL). Following concentration in vacuo, the residue was puri-

fied by flash chromatography (silica gel, 10% ethyl acetate in

hexanes) to afford E-28 (0.760 g, 2.43 mmol, 99% yield, >99%

1

E) as a clear, colorless oil. H NMR (400 MHz, CDCl₃) δ 0.87

(3H, t, J₁) 6.9 Hz), 1.20-1.70 (23H, m), 2.00 (2H, q, J₁) 6.8

Hz), 3.30-3.85 (5H, m), 4.54 (1H, t, J₁) 2.0 Hz), 5.41 (1H, d

of d of t, J₁) 15.4 Hz, J₂) 7.1 Hz, J₃) 1.1), 5.60 (1H, t of t

of d, J₁) 15.4 Hz, J₂) 6.7 Hz, J₃) 0.9 Hz). ¹³C NMR (100

MHz, CDCl₃) δ 9.8, 14.0, 19.7, 25.5, 26.2, 27.8, 29.1, 29.4, 29.5,

29.7, 30.1, 30.8, 36.6, 62.3, 67.7, 74.5, 98.8, 132.4, 132.7. GC/

MS: 283 (0.1), 199 (0.7), 135 (0.9), 123 (1.5), 109 (5.0), 101

(20.5), 85 (100), 41 (48.3). Anal. Calcd. for C₁₉H₃₆O₃: C, 73.03;

H, 11.61. Found: C, 72.98; H, 11.61.

9-(2-(1-Hydroxypropyl)-Z-cyclopropyl)-1-(tetrahydro-

pyran-2′-yloxy)-nonane (Z-29). Procedure D, 100% yield

from Z-28. ¹H NMR (400 MHz, CDCl₃) δ -0.01 (1H, d of d, J₁

) 5.2 Hz, J₂) 5.2 Hz), 0.66 (1H, t of d of d, J₁) 8.4 Hz, J₂)

4.4 Hz, J₃) 0.4 Hz), 0.75-0.85 (2H, m), 0.95 (3H, t, J₁) 7.6

Hz), 1.20-1.80 (25H, m), 3.08 (1H, d of t, J₁) 8.1 Hz, J₂)

6.2 Hz), 3.30-3.85 (4H, m), 4.53 (1H, t, J₁) 2.8 Hz). ¹³C NMR

(100 MHz, CDCl₃) δ 9.4, 9.9, 16.8, 19.6, 22.8, 25.4, 26.2, 29.1,

29.40, 29.43, 29.49, 29.50, 29.7, 30.1, 30.7, 36.6, 62.2, 67.6, 73.7,

98.7. GC/MS: 213 (7.2), 121 (1.2), 109 (2.6), 101 (14.1), 95 (7.4),

85 (100), 57 (30.0), 41 (51.6). Anal. Calcd. for C₂₀H₃₈O₃: C,

73.57; H, 11.73. Found: C, 73.23; H, 11.95.

9-(2-(1-Hydroxypropyl)-E-cyclopropyl)-1-(tetrahydro-

pyran-2′-yloxy)-nonane (E-29). Procedure D, 98% yield from

E-28. ¹H NMR (400 MHz, CDCl₃) δ 0.23 (1H, m), 0.38 (1H,

m), 0.58 (2H, m), 0.95 (3H, t, J₁) 7.4 Hz, 1.15-1.80 (25H,

m), 2.79 (1H, q of d, J₁) 6.9 Hz, J₂) 1.2 Hz), 3.30-3.90 (4H,

12,12′-Dimethoxydodecanal (30). Into a solution of cy-

clododecene (3.000 g, 18.07 mmol) in 20% methanol in CH₂-

2466 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

Cl₂(40 mL) being stirred at -40 °C was bubbled excess ozone

gas. After the ozonide had formed, the reaction was purged

with oxygen and nitrogen before p-TsOH (0.350 g, 10% w/w)

was added, and the reaction was allowed to warm to room

temperature. After being stirred at room temperature for 90

min, solid NaHCO₃(2.000 g) was added followed 15 min later

by dimethyl sulfide (4.00 mL), and the reaction was stirred at

room temperature overnight under nitrogen. Brine (80 mL)

was added, and the solution was extracted with CH₂Cl₂(2 ×

60 mL) following separation of the layers. The organic layers

were washed with brine (80 mL) and dried over MgSO₄, and

the solvent removed in vacuo to afford 30 (3.730 g, 16.59 mmol,

92% yield) as a clear, colorless oil. ¹H NMR (200 MHz, CDCl₃)

δ 1.10-1.30 (14H, m), 1.59 (4H, m), 2.35 (2H, t of d, J₁) 7.3

Hz, J₂) 1.8 Hz), 3.23 (6H, s), 4.28 (1H, t, J₁) 5.6 Hz), 9.68

(1H, t, J₁) 1.8 Hz). ¹³C NMR (50 MHz, CDCl₃) δ 21.9, 24.4,

29.0, 29.20, 29.24, 29.31, 29.32, 29.4, 32.3, 43.8, 52.4 (2C),

104.4, 202.7. GC/MS: 213 (2.9), 163 (0.8), 121 (1.4), 95 (5.0),

75 (100). Anal. Calcd. for C₁₄H₂₈O₃: C, 69.08; H, 11.21.

Found: C, 68.81; H, 11.55.

Hz, J₂) 12.0 Hz, J₃) 7.7 Hz, J₄) 3.9 Hz), 0.86 (1H, d of d

of d of d, J₁) 16.3 Hz, J₂) 8.4 Hz, J₃) 5.1 Hz, J₄) 5.1 Hz),

1.20-1.45 (16H, m), 1.54 (2H, t, J₁) 6.7 Hz), 1.66 (1H, br s),

2.15 (2H, d of d of d, J₁) 7.0 Hz, J₂) 6.4 Hz, J₃) 1.5 Hz),

2.80 (1H, d of t, J₁) 8.5 Hz, J₂) 6.1 Hz), 3.69 (3H, s), 5.77

(1H, d of t, J₁) 15.7 Hz, J₂) 1.5 Hz), 6.94 (1H, d of t, J₁)

15.7 Hz, J₂) 6.9 Hz). ¹³C NMR (50 MHz, CDCl₃) δ 2.4, 2.7,

18.0, 25.7, 27.9, 29.0, 29.3, 29.40, 29.43, 29.5, 29.7, 32.2, 37.2,

51.4, 76.9, 120.7, 149.9, 167.2. GC/MS: 247 (0.3), 226 (1.8),

152 (5.4), 129 (14.4), 43 (100). Anal. Calcd. for C₁₈H₃₂O₃: C,

71.60; H, 10.52. Found: C, 71.22; H, 10.90.

14-Cyclopropyl-14-hydroxytetradecanoic Acid (8a).

Procedures C and G, 55% yield from 34, mp 78.0-79.0 °C. ¹H

NMR (400 MHz, CDCl₃) δ 0.20 (2H, d of d of d of d, J₁) 22.0

Hz, J₂) 13.1 Hz, J₃) 4.9 Hz, J₄) 4.1 Hz), 0.48 (2H, d of d

of d of d, J₁) 17.2 Hz, J₂) 12.0 Hz, J₃) 7.7 Hz, J₄) 3.9 Hz),

0.87 (1H, d of d of d of d, J₁) 16.3 Hz, J₂) 8.4 Hz, J₃) 5.1

Hz, J₄) 5.1 Hz), 1.20-1.45 (20H, m), 1.58 (2H, t of d, J₁)

6.3 Hz, J₂) 1.5 Hz), 1.61 (2H, quint, J₁) 7.3 Hz), 2.32 (2H,

t, J₁) 7.5 Hz), 2.83 (1H, d of t, J₁) 8.5 Hz, J₂) 6.4 Hz). ¹³

C

14,14′-Dimethoxytetradec-1-en-3-ol (31). Procedure J,

NMR (100 MHz, CDCl₃) δ 2.4, 2.8, 17.9, 24.6, 25.6, 28.9, 29.1,

29.25, 29.33, 29.40, 29.43, 29.47, 29.6, 34.0, 37.1, 77.1, 179.4.

Anal. Calcd. for C₁₇H₃₂O₃: C, 71.79; H, 11.34. Found: C, 71.73;

H, 11.70.

1

92% yield from 30. H NMR (200 MHz, CDCl₃) δ 1.10-1.40

(16H, m), 1.51 (4H, m), 1.80 (1H, br s), 3.26 (6H, s), 4.03 (1H,

q, J₁) 6.3 Hz), 4.31 (1H, t, J₁) 5.6 Hz), 5.10 (2H, m), 5.82

(1H, m). ¹³C NMR (50 MHz, CDCl₃) δ 24.5, 25.3, 29.3, 29.38,

29.45, 29.46, 29.48, 29.50, 32.4, 37.0, 52.5 (2C), 73.1, 104.5,

114.4, 141.3. GC/MS: 223 (0.8), 152 (1.0), 109 (4.2), 95 (8.0),

75 (100). Anal. Calcd. for C₁₆H₃₂O₃: C, 70.54; H, 11.84.

Found: C, 70.51; H, 11.56.

Methyl 14-Cyclopropyl-14-hydroxytetradecanoate (8).

Prepared by derivatization of 8a with an ethereal solution of

diazomethane. GC/MS: 280 (0.2), 228 (16.1), 185 (10.7), 143

(17.4), 43 (100).

1-(Tetrahydropyran-2′-yloxy)-10-(hydroxymethyl)-tet-

radec-11-ene (35). To a solution of diisopropylamine (355 µL,

2.58 mmol) in THF (10 mL) being stirred at -5 °C under a

nitrogen atmosphere was added n-butyllithium (1.54 mL, 2.37

mmol, 1.5M in hexanes). After 30 min, Z-hex-2-enal-N,N′-

dimethyl hydrazone³⁰(0.300 g, 2.15 mmol) was added, and 1

h later, 9-bromo-1-(tetrahydropyran-2′-yloxy)-nonane⁴⁹(0.650

g, 2.37 mmol) was added. The reaction was left to stir for 12

h before being quenched by water (50 mL). CuCl₂(0.310 g,

2.37 mmol) was added, and the reaction was stirred for 1 h.

The reaction mixture was then quenched by addition of

aqueous, 3 N NH₄OH (30 mL) and extracted with ethyl acetate

(3 × 20 mL), and the combined organic layers were washed

with brine (30 mL), dried over Na₂SO₄, and concentrated in

vacuo. The residue was dissolved in methanol (20 mL) and

sodium borohydride (0.050 g, 6.00 mmol) added with stirring

under a nitrogen atmosphere. After 2 h, the reaction mixture

was quenched by addition of aqueous, 5% oxalic acid (50 mL)

and extracted with CH₂Cl₂(3 × 30 mL). The combined organic

layers were washed with brine (30 mL), dried over Na₂SO₄,

and concentrated in vacuo. The residue was purified using

flash chromatography (silica gel, 10% ethyl acetate in hexanes)

to afford 35 (0.135 g, 0.43 mmol, 20% yield over three steps,

75% E isomer) as a clear, colorless oil. ¹H NMR (400 MHz,

CDCl₃, E and Z isomers) δ 0.95 (3H, t, J₁) 7.5 Hz), 1.20-

1.80 (23H, m), 2.06 (2H, m), 2.34 (1H, m), 3.25-3.90 (6H, 4m)

4.54 (1H, t, J₁) 3.5 Hz), 5.03 (0.75H, d of d of t, J₁) 21 Hz,

J₂) 8.6 Hz, J₃) 1.5 Hz), 5.11 (0.25H, d of d of t, J₁) 8.8 Hz,

J₂) 7.7 Hz, J₃) 1.5 Hz), 5.56 (1H, m). ¹³C NMR (100 MHz,

CDCl₃, E isomer) δ 14.5, 19.7, 21.1, 25.5, 26.2, 27.1, 29.43,

29.47, 29.52, 29.68, 29.72, 30.8, 31.4, 40.4, 62.3, 66.5, 67.7, 98.8,

130.4, 135.2. GC/MS (hydrazone): 366 (M^+•, 4.1), 337 (2.3),

281 (8.4), 238 (9.8), 139 (64.1), 101 (2.3), 85 (93.0), 41 (100).

GC/MS (aldehyde): 324 (M^+•, 0.8), 294 (0.6), 272 (0.6), 240

(1.3), 123 (1.1), 101 (11.9), 85 (100), 41 (61.8). GC/MS (alco-

hol): 297 (0.6), 224 (1.1), 149 (1.0), 123 (2.0), 109 (3.6), 101

(8.0), 85 (100), 41 (58.3). Anal. Calcd. for C₂₀H₃₈O₃: C, 73.57;

H, 11.73. Found: C, 73.37; H, 11.82.

1-Cyclopropyl-12,12′-dimethoxydodecan-1-ol (32). Pro-

cedure D, 92% yield from 31. ¹H NMR (200 MHz, CDCl₃) δ

0.21 (2H, d of d of d of d, J₁) 22.0 Hz, J₂) 13.1 Hz, J₃) 4.9

Hz, J₄) 4.1 Hz), 0.48 (2H, d of d of d of d, J₁) 17.2 Hz, J₂)

12.0 Hz, J₃) 7.7 Hz, J₄) 3.9 Hz), 0.87 (1H, d of d of d of d,

J₁) 16.3 Hz, J₂) 8.4 Hz, J₃) 5.1 Hz, J₄) 5.1 Hz), 1.20-

1.60 (21H, m), 2.82 (1H, d of t, J₁) 8.4 Hz, J₂) 6.4 Hz), 3.29

(6H, s), 4.33 (1H, t, J₁) 2.9 Hz). ¹³C NMR (50 MHz, CDCl₃) δ

2.4, 2.8, 17.9, 22.0, 24.5, 25.7, 29.1, 29.37, 29.39, 29.5, 29.7,

32.4, 37.2, 52.5 (2C), 76.9, 104.4. GC/MS: 286 (M^+•, 0.2), 223

(0.5), 152 (2.3), 109 (2.7), 75 (100). Anal. Calcd. for C₁₇H₃₄O₃:

C, 71.28; H, 11.96. Found: C, 71.13; H, 11.94.

12-Cyclopropyl-12-hydroxydodecanal (33). Procedure B,

1

97% yield from 32. H NMR (200 MHz, CDCl₃) δ 0.20 (2H, d

of d of d of d, J₁) 22.0 Hz, J₂) 13.1 Hz, J₃) 4.9 Hz, J₄) 4.1

Hz), 0.47 (2H, d of d of d of d, J₁) 17.2 Hz, J₂) 12.0 Hz, J₃

) 7.7 Hz, J₄) 3.9 Hz), 0.85 (1H, d of d of d of d, J₁) 16.3 Hz,

J₂) 8.4 Hz, J₃) 5.1 Hz, J₄) 5.1 Hz), 1.20-1.60 (18H, m),

1.86 (1H, br s), 2.39 (2H, t of d, J₁) 3.7 Hz, J₂) 0.9 Hz), 2.81

(1H, d of t, J₁) 8.4 Hz, J₂) 6.4 Hz), 9.73 (1H, s). ¹³C NMR

(50 MHz, CDCl₃) δ 2.4, 2.8, 18.0, 22.0, 25.7, 29.1, 29.31, 29.37,

29.49, 29.54, 29.7, 37.2, 43.9, 77.2, 203.0. GC/MS: 286 (M^+•

,

0.2), 223 (0.5), 152 (2.3), 109 (2.7), 75 (100). Anal. Calcd. for

C₁₅H₂₈O₂: C, 74.95; H, 11.74. Found: C, 74.97; H, 12.12.

12-Cyclopropyl-12-hydroxydodecanoic Acid (6a). Pro-

cedures E and I, 62% yield from 33, mp 62.0-63.0 °C. ¹H NMR

(400 MHz, CDCl₃) δ 0.20 (2H, d of d of d of d, J₁) 22.0 Hz, J₂

) 13.1 Hz, J₃) 4.9 Hz, J₄) 4.1 Hz), 0.48 (2H, d of d of d of

d, J₁) 17.2 Hz, J₂) 12.0 Hz, J₃) 7.7 Hz, J₄) 3.9 Hz), 0.87

(1H, d of d of d of d, J₁) 16.3 Hz, J₂) 8.4 Hz, J₃) 5.1 Hz, J₄

) 5.1 Hz), 1.20-1.55 (20H, m), 2.32 (2H, t, J₁) 7.5 Hz), 2.83

(1H, d of t, J₁) 8.4 Hz, J₂) 6.4 Hz). ¹³C NMR (100 MHz,

CDCl₃) δ 2.4, 2.8, 17.9, 24.6, 25.6, 28.9, 29.1, 29.3, 29.4, 29.5,

29.6, 34.0, 37.1, 77.1, 179.4. Anal. Calcd. for C₁₅H₂₈O₃: C,

70.27; H, 11.01. Found: C, 70.23; H, 11.31.

Methyl 12-Cyclopropyl-12-hydroxydodecanoate (6).

Prepared by derivatization of 6a with an ethereal solution of

diazomethane. GC/MS: 237 (0.2), 200 (15.0), 157 (12.9), 143

(17.7), 43 (100).

Methyl 14-Cyclopropyl-14-hydroxytetradec-2-enoate

(34). Procedure F, 79% yield from 33. ¹H NMR (200 MHz,

CDCl₃) δ 0.18 (2H, d of d of d of d, J₁) 22.0 Hz, J₂) 13.1 Hz,

J₃) 4.9 Hz, J₄) 4.1 Hz), 0.46 (2H, d of d of d of d, J₁) 17.2

1-(Tetrahydropyran-2′-yloxy)-10-(t-butyldiphenylsi-

loxymethyl)-tetradec-11-ene (36). To a solution of 35 (0.040

g, 0.127 mmol) in DMF (2 mL) being stirred at room temper-

(49) Sharma, A.; Chattopadhyay, S. J. Org. Chem. 1998, 63, 6128-

6131.

J. Org. Chem, Vol. 70, No. 7, 2005 2467

Cryle et al.

ature was added TBDPSCl (43 µL, 0.153 mmol) and imidazole

(0.020 g, 0.254 mmol). After 10 min, the reaction was quenched

by the addition of water (10 mL)/diethyl ether (10 mL), and

the aqueous phase was extracted with diethyl ether (10 mL).

The combined organic layers were washed with brine (20 mL),

dried over Na₂SO₄, and concentrated in vacuo. The residue was

purified using flash chromatography (silica gel, 5% ethyl

acetate in hexanes) to afford 36 (0.0453 g, 0.0828 mmol, 65%

yield, 75% E isomer) as a clear, colorless oil. ¹H NMR (400

MHz, CDCl₃, E and Z isomers) δ 0.90 (3H, t, J₁) 7.5 Hz),

1.04 (9H, s), 1.20-1.60 (20H, m), 1.70-1.85 (2H, 2m), 1.95 (2H,

m), 2.52 (1H, m), 3.35-3.90 (6H, 4m) 4.57 (1H, t, J₁) 2.7 Hz),

5.06 (0.75H, d of d of t, J₁) 21.0 Hz, J₂) 8.4 Hz, J₃) 1.5

Hz), 5.11 (0.25H, d of d of t, J₁) 8.8 Hz, J₂) 8.3 Hz, J₃) 1.5

Hz), 5.56 (1H, m), 7.38 (6H, m), 7.66 (4H, m). ¹³C NMR (100

MHz, CDCl₃, E isomer) δ 14.5, 19.7 (3C), 21.0, 25.5, 26.2, 26.9,

27.1, 29.49, 29.53, 29.6, 29.68, 29.72, 29.8, 30.8, 31.6, 40.2, 62.3,

66.6, 67.7, 98.8, 127.5 (4C), 129.4 (2C), 131.0, 132.8, 134.0 (2C),

135.6 (4C). Anal. calcd. for C₃₆H₅₆O₃: C, 76.55; H, 9.99.

Found: C, 76.33; H, 10.21.

109 (2.1), 101 (12.9), 85 (98.9), 41 (100). Anal. Calcd. for

C₁₈H₃₄O₃: C, 72.44; H, 11.48. Found: C, 72.59; H, 11.59.

1-(Tetrahydropyran-2′-yloxy)-10-hydroxypentadec-12-

1

ene (40). Procedures K and L, 72% yield from 39. H NMR

(400 MHz, CDCl₃) δ 0.95 (3H, t, J₁) 7.5 Hz), 1.20-1.85 (23H,

m), 2.05 (2H, m), 2.19 (2H, m), 3.30-3.50 (2H, 2m), 3.55 (1H,

m), 3.65-3.85 (2H, 2m), 4.55 (1H, t, J₁) 3.5 Hz), 5.36 (1H,

m), 5.56 (1H, m). ¹³C NMR (100 MHz, CDCl₃) δ 14.2, 19.7, 20.7,

25.5, 25.7, 26.2, 29.4, 29.5, 29.6, 29.7, 30.8, 35.2, 36.7, 40.6,

62.3, 67.7, 71.5, 98.8, 124.5, 135.1. GC/MS: 283 (0.2), 224 (0.7),

173 (4.0), 109 (1.9), 101 (10.7), 85 (100), 43 (23.7), 41 (38.7).

Anal. Calcd. for C₂₀H₃₈O₃: C, 73.57; H, 11.73. Found: C, 73.77;

H, 11.67.

Pentadec-12-en-1,10-diol (41). Procedure B, 90% yield

1

from 40. H NMR (400 MHz, CDCl₃) δ 0.95 (3H, t, J₁) 7.5

Hz), 1.20-1.60 (18H, m), 2.05 (2H, m), 2.20 (2H, m), (3.68,

1H, quint, J₁) 5.8 Hz), 3.62 (2H, t, J₁) 6.6 Hz), 5.36 (1H,

m), 5.55 (1H, m). ¹³C NMR (100 MHz, CDCl₃) δ 14.2, 20.7, 25.7,

29.4, 29.5, 29.6, 29.7, 32.8, 35.2, 36.8, 40.6, 63.1, 71.5, 124.5,

135.1. Anal. Calcd. for C₁₅H₃₀O₂: C, 74.33; H, 12.41. Found:

C, 74.27; H, 12.10.

Methyl 10-(t-Butyldiphenylsiloxymethyl)-tetradec-11-

enoate (37). Procedures B and E, 80% yield from 36, 75% E

Methyl 10-Oxopentadec-12-enoate (42). Procedure E,

esterification with ethereal diazomethane, 88% yield from 41.

¹H NMR (400 MHz, CDCl₃) δ 0.95 (3H, t, J₁) 7.5 Hz), 1.20-

1.60 (12H, m), 2.03 (2H, quint, J₁) 7.4 Hz), 2.27 (2H, t, J₁)

7.9 Hz), 2.39 (2H, t, J₁) 7.4 Hz), 3.12 (2H, d, J₁) 6.9 Hz),

3.64 (3H, s), 5.51 (2H, m). ¹³C NMR (100 MHz, CDCl₃) δ 13.9,

20.8, 23.7, 24.9, 29.05, 29.11, 29.2, 29.7, 34.1, 41.6, 42.3, 51.4,

121.0, 135.2, 174.3, 209.3. GC/MS: 268 (M^+•, 1.0), 237 (3.5),

200 (6.0), 199 (49.9), 139 (29.8), 121 (21.8), 41 (100). Anal.

Calcd. for C₁₆H₂₈O₃: C, 71.60; H, 10.51. Found: C, 71.44; H,

10.74.

1

isomer. H NMR (400 MHz, CDCl₃, E and Z isomers) δ 0.89

(3H, t, J₁) 7.5 Hz), 1.03 (9H, s), 1.10-1.70 (14H, m), 1.97

(2H, m), 2.28 (2H, t, J₁) 7.6 Hz), 2.50 (1H, m), 3.48 (2H, d, J₁

) 6.4 Hz), 3.65 (3H, s), 5.05 (0.75H, m), 5.19 (0.25H, m), 5.42

(1H, m), 7.38 (6H, m), 7.66 (4H, m). ¹³C NMR (100 MHz, CDCl₃,

E isomer) δ 14.5, 19.3 (3C), 21.0, 25.0, 26.9, 27.0, 29.1, 29.4,

29.7, 30.3, 31.6, 34.1, 40.0, 51.4, 67.6, 127.5 (4C), 129.5 (2C),

130.9, 132.9, 134.0 (2C), 135.6 (4), 174.3. Anal. Calcd. for

C₃₂H₄₈O₃Si: C, 75.54; H, 9.51. Found: C, 75.40; H, 9.58.

HRMS: calcd for C₃₂H₄₈O₃SiNa, 531.3271; found, 531.3273.

Methyl 10-Hydroxymethyltetradec-11-enoate (11). To

a solution of 37 (0.015 g, 0.0308 mmol) in THF (20 mL) being

stirred at 0 °C was added TBAF (23 µL, 1.0 M in THF, 0.0370

mmol) and acetic acid (1 µL, 0.0308 mmol), and the reaction

was stirred at room temperature for 30 h before being

quenched by addition of brine (20 mL). Following separation

of the layer, the aqueous phase was extracted with diethyl

ether (3 × 10 mL), the combined organic layers were dried

over Na₂SO₄, and the solvent was removed in vacuo. The

residue was purified using flash chromatography (silica gel,

10% ethyl acetate in hexanes) to afford 11 (0.008 g, 0.0308

Methyl 10-Hydroxypentadec-12-enoate (13). Procedure

1

I, 75% yield from 42, mp 34.0-35.0 °C. H NMR (400 MHz,

CDCl₃) δ 0.95 (3H, t, J₁) 7.5 Hz), 1.20-1.60 (15H, m), 2.03

(2H, d of t, J₁) 7.4 Hz, J₂) 6.0 Hz), 2.18 (2H, d of t, J₁) 7.3

Hz, J₂) 6.3 Hz), 2.27 (2H, t, J₁) 7.5 Hz), 3.58 (1H, m), 3.64

(3H, s), 5.36 (1H, m), 5.55 (1H, m). ¹³C NMR (100 MHz, CDCl₃)

δ 14.2, 20.7, 24.9, 25.7, 29.1, 29.2, 29.6, 34.1, 35.2, 36.7, 40.7,

51.4, 71.4, 124.5, 135.1, 174.3. GC/MS: 252 (0.3), 201 (15.4),

170 (8.5), 169 (78.8), 151 (4.7), 133 (8.5), 55 (98.7), 41 (100).

Anal. Calcd. for C₁₆H₃₀O₃: C, 71.07; H, 11.18. Found: C, 70.93;

H, 11.44.

1-(Tetrahydropyran-2′-yloxy)-tridec-10-en-13-ol (43).

Procedure L, 86% yield from 38. ¹H NMR (400 MHz, CDCl₃) δ

1.20-1.80 (21H, m), 1.98 (1H, q, J₁) 6.9 Hz), 2.03 (1H, q, J₁

) 6.8 Hz), 2.23 (1H, q of q, J₁) 7.0 Hz, J₂) 1.0 Hz), 2.30

(1H, q of q, J₁) 7.2 Hz, J₂) 0.6 Hz), 3.30-3.50 (2H, 2m),

3.60 (2H, br d, J₁) 4.4 Hz), 3.65-3.90 (2H, 2m), 4.55 (1H, t,

J₁) 3.5 Hz), 5.35 (1H, m), 5.52 (1H, m). ¹³C NMR (100 MHz,

CDCl₃, E & Z isomers) δ 19.7, 25.5, 26.2, 27.3, 29.1, 29.38,

29.40, 29.42, 29.5, 29.6, 29.7, 30.76, 30.78, 36.0, 62.0, 62.3, 67.7,

98.8, 124.9, 125.7, 133.5, 134.3. GC/MS: 297 (0.2), 268 (0.5),

196 (1.3), 137 (1.0), 101 (15.0), 85 (100), 55 (34.2), 41 (48.8).

Anal. Calcd. for C₁₈H₃₄O₃: C, 72.44; H, 11.48. Found: C, 72.31;

H, 11.59.

13-(Tetrahydropyran-2′-yloxy)-tridec-3-enal (44). Pro-

cedure H, 94% yield from 43. ¹H NMR (400 MHz, CDCl₃) δ

1.20-1.80 (20H, m), 2.02 (2H, 2t, J₁) 7.0 Hz), 3.08 (1H, d of

m, J₁) 7.2 Hz), 3.16 (1H, d of m, J₁) 7.2 Hz), 3.30-3.90 (4H,

4m), 4.55 (1H, t, J₁) 3.5 Hz), 5.42-5.72 (2H, m), 9.63 (1H, d

of t, J₁) 2.5 Hz, J₂) 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃, E

and Z isomers) δ 19.7, 25.5, 26.2, 27.6, 29.1, 29.39, 29.41, 29.44,

29.5, 29.7, 30.8, 32.7, 42.6, 47.3, 62.4, 67.7, 98.9, 117.9, 119.0,

135.5, 137.0, 199.8, 200.4. GC/MS: 296 (M^+•, 0.1), 177 (0.4),

135 (1.7), 121 (4.0), 101 (53.5), 85 (100), 55 (25.5), 41 (67.2).

Anal. Calcd. for C₁₈H₃₂O₃: C, 72.93; H, 10.88. Found: C, 72.55;

H, 10.80.

1

mmol, 100% yield, 75% E isomer) as a clear, colorless oil. H

NMR (400 MHz, CDCl₃, E and Z isomers) δ 0.95 (3H, t, J₁)

7.5 Hz), 1.20-1.60 (15H, m), 2.06 (2H, m), 2.28 (2H, t, J₁)

7.5 Hz), 2.57 (1H, m), 3.30, (1H, t, J₁) 9.3 Hz), 3.52 (1H, m),

3.64 (3H, s), 5.01 (0.75H, d of d of t, J₁) 21.0 Hz, J₂) 8.4 Hz,

J₃) 1.5 Hz), 5.11 (0.25H, d of d of t, J₁) 8.8 Hz, J₂) 8.3 Hz,

J₃) 1.5 Hz), 5.59 (1H, d of t, J₁) 11.7 Hz, J₂) 7.4 Hz). ¹³C

NMR (100 MHz, CDCl₃, E isomer) δ 14.5, 21.1, 24.9, 27.1, 29.1,

29.2, 29.3, 29.6, 31.4, 34.1, 40.4, 51.4, 66.5, 134.0, 135.6, 174.3.

GC/MS: 252 (M^+•- H₂O, 2.0), 240 (8.5), 208 (23.0), 166 (16.4),

123 (16.3), 109 (22.9), 69 (70.7), 55 (100). Anal. Calcd. for

C₁₆H₃₀O₃: C, 71.07; H, 11.18. Found: C, 71.07; H, 11.18.

10-(Tetrahydropyran-2′-yloxy)-decanal (38). Procedure

K, 75% yield from 1-(tetrahydropyran-2′-yloxy)-undec-10-ene.¹⁸

¹H NMR (400 MHz, CDCl₃) δ 1.20-1.80 (22H, m), 3.30-3.90

(4H, 4m), 4.53 (1H, t, J₁) 3.5 Hz), 9.72 (1H, t, J₁) 1.8 Hz).

¹³C NMR (100 MHz, CDCl₃) δ 19.6, 22.0, 25.4, 26.1, 29.2, 29.3,

29.4, 29.5, 29.6, 30.7, 43.8, 62.3, 67.6, 98.8, 202.9. GC/MS: 256

(M^+•, 0.1), 255 (0.7), 183 (0.5), 155 (1.1), 137 (1.8), 101 (35.2),

85 (100), 41 (71.9). Anal. Calcd. for C₁₅H₂₈O₃: C, 70.27; H,

11.01. Found: C, 70.10; H, 11.25.

1-(Tetrahydropyran-2′-yloxy)-tridec-12-en-10-ol (39).

Procedure J, 87% yield from 38. ¹H NMR (400 MHz, CDCl₃) δ

1.20-1.80 (23H, m), 2.02-2.28 (2H, 2m), 3.30-3.50 (2H, 2m),

3.57 (1H, t of t, J₁) 6.7 Hz, J₂) 5.4 Hz), 3.65-3.85 (2H, 2m),

4.53 (1H, t, J₁) 3.5 Hz), 5.06 (1H, m), 5.09 (1H, m), 5.79 (1H,

m). ¹³C NMR (100 MHz, CDCl₃) δ 19.6, 25.4, 25.6, 26.1, 29.36,

29.43, 29.45, 29.54, 29.7, 30.7, 36.7, 41.9, 62.2, 67.6, 70.6, 98.7,

117.8, 134.9. GC/MS: 297 (0.2), 281 (0.5), 173 (1.9), 137 (2.6),

1-(Tetrahydropyran-2′-yloxy)-tetradec-10-en-13-ol (45).

Procedure J, 87% yield from 44. ¹H NMR (400 MHz, CDCl₃) δ

1.15 (1.5H, d, J₁) 6.2 Hz), 1.18 (1.5H, d, J₁) 6.2 Hz), 1.20-

1.80 (21H, m), 1.95-2.25 (4H, 2m), 3.30-3.50 (2H, 2m), 3.65-

2468 J. Org. Chem., Vol. 70, No. 7, 2005

Fatty Acids as Mechanistic Probes for Cytochrome P450

3.85 (3H, 3m), 4.55 (1H, t, J₁) 3.5 Hz), 5.38 (1H, m), 5.55

(1H, m). ¹³C NMR (100 MHz, CDCl₃, E and Z isomers) δ 19.7,

22.6, 22.7, 25.5, 26.2, 27.4, 29.1, 29.3, 29.39, 29.41, 29.43, 29.5,

29.7, 30.8, 32.6, 37.1, 42.5, 62.3, 67.2, 67.7 (2C), 98.8, 125.0,

125.7, 133.5, 134.8. GC/MS: 281 (0.5), 210 (1.1), 195 (1.0), 123

(1.7), 101 (13.3), 85 (100), 45 (35.9), 41 (36.3). Anal. Calcd. for

C₁₉H₃₆O₃: C, 73.03; H, 11.61. Found: C, 73.06; H, 11.30.

Tetradec-10-en-1,13-diol (46). Procedure B, 91% yield

from 45. ¹H NMR (400 MHz, CDCl₃) δ 1.16 (1.5H, d, J₁) 6.2

Hz), 1.19 (1.5H, d, J₁) 6.2 Hz), 1.20-1.60 (16H, m), 1.98-

2.10 (3H, m), 2.30 (1H, m), 3.62 (2H, t, J₁) 6.6 Hz), 3.78 (1H,

m), 5.38 (1H, m), 5.55 (1H, m). ¹³C NMR (100 MHz, CDCl₃, E

& Z isomers) δ 22.6, 22.8, 25.7, 27.4, 29.1, 29.2, 29.36, 29.41,

29.48, 29.50, 29.6, 29.7, 32.6, 32.8, 37.1, 42.5, 63.1, 67.2, 67.7,

125.0, 125.7, 133.5, 134.8. Anal. Calcd. For C₁₄H₂₈O₂: C, 73.63;

H, 12.36. Found C, 73.72; H, 12.23.

(2-Hydroxypropanol)-9-cyclopropylnonan-1-ol (47). Pro-

cedure D, 89% yield from 46. ¹H NMR (400 MHz, CDCl₃) δ

-0.25 (0.5H, m), 0.22 (1H, m), 0.43 (1H, m), 0.68 (1.5H, m),

1.18 (1.5H, d, J₁) 6.4 Hz), 1.22 (1.5H, d, J₁) 6.4 Hz), 1.25-

1.70 (20H, m), 3.61 (2H, t, J₁) 6.6 Hz), 3.86 (1H, m). ¹³C NMR

(100 MHz, CDCl₃, E and Z isomers) δ 10.9, 11.7, 12.3, 14.7,

15.1, 15.2, 18.1, 23.1, 25.7, 28.9, 29.37, 29.39, 29.44, 29.5, 29.55,

29.56, 29.7, 30.0, 32.8, 38.0, 38.1, 43.7, 63.1, 68.8, 69.1.

HRMS: calcd for C₁₅H₃₀O₂, 242.2246; found, 242.2248. Anal.

Calcd. for C₁₅H₂₈O₂: C, 74.32; H, 12.47. Found: C, 74.67; H,

12.10.

12.2, 12.3, 14.7, 15.1, 15.2, 15.4, 18.1, 18.7, 23.1, 23.3, 24.9,

28.9, 29.0, 29.1, 29.2, 29.4, 29.5, 29.6, 29.7, 30.0, 34.10, 34.11,

34.2, 38.0, 38.1, 43.70, 43.72, 51.4, 68.6, 68.75, 68.81, 69.0,

174.4. GC/MS: 270 (M^+•, 0.1), 252 (0.4), 220 (1.0), 194 (2.3),

178 (1.7), 152 (3.5), 55 (67.3), 45 (100). Anal. Calcd. for

C₁₆H₃₀O₃: C, 71.07; H, 11.18. Found: C, 70.89; H, 11.21.

Methyl 15-Hydroxypentadec-12-ene (15). Procedure L,

60% yield from methyl 12-oxododecanoate,³²75% E isomer,

mp 44.0-45.0 °C. ¹H NMR (400 MHz, CDCl₃, E isomer) δ

1.20-1.35 (14H, m), 1.57 (2H, quint, J₁) 7.5 Hz), 1.68 (1H,

br s), 1.95 (2H, d of t, J₁) 6.9 Hz, J₂) 6.7 Hz), 2.22 (2H, d of

d of t, J₁) 6.0 Hz, J₂) 0.90 Hz, J₃) 7.4 Hz), 2.25 (2H, t, J₁

) 7.5 Hz), 3.57 (2H, t, J₁) 6.4 Hz), 3.61 (3H, s), 5.33 (1H, m),

5.49 (1H, m). ¹³C NMR (100 MHz, CDCl₃, E-isomer) δ 24.9,

29.07, 29.16, 29.19, 29.33, 29.37, 29.44, 29.6, 30.7, 34.0, 35.9,

51.4, 62.0, 125.7, 134.2, 174.3. GC/MS: 252 (1.6), 240 (2.7),

208 (9.6), 166 (6.8), 124 (8.5), 110 (11.2), 55 (94.0), 41 (100).

Anal. Calcd. for C₁₆H₃₀O₃: C, 71.07; H, 11.18. Found: C, 70.73;

H, 11.54.

Acknowledgment. M.J.C. is grateful to the Uni-

versity of Queensland Graduate School for the Graduate

School Travel Award and the University of Queensland

Department of Chemistry for the Francine Kroesen

Scholarship.

Supporting Information Available: General methods

and instrumentation; experimental procedures for 1a, 2a,

E-3a, 4a-8a, 1-9, 13, 15, 19, Z-20, 21, 23-27, Z-28, 29, 31-

34, and 37-47; GC conditions for turnover analysis; retention

times of synthesized standards; and NMR determination of

rearrangement product from E-1a. This information is avail-

able free of charge via the Internet at http://pubs.acs.org.

Methyl (2-Hydroxypropanol)-9-cyclopropylnonanoate

(9). Procedure E, esterification with ethereal diazomethane,

procedure I, 90% yield from 47, mp 39.0-40.0 °C. ¹H NMR

(400 MHz, CDCl₃, E and Z isomers) δ -0.25 (0.5H, m), 0.22

(1H, m), 0.43 (1H, m), 0.68 (1.5H, m), 1.18 (1.5H, d, J₁) 6.4

Hz), 1.22 (1.5H, d, J₁) 6.4 Hz), 1.25-1.80 (17H, m), 2.28 (2H,

t, J₁) 7.5 Hz), 3.64 (3H, s), 3.87 (1H, m). ¹³C NMR (100 MHz,

CDCl₃, E and Z plus syn/anti isomers) δ 10.8, 10.9, 11.2, 11.7,

JO047985D

J. Org. Chem, Vol. 70, No. 7, 2005 2469

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Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

DOI: 10.1021/jo047985d

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