Studies on estrogen bios~thesis using radioactive and stable isotopes J. Neville Wright and Muhammad Akhtar

ofBiochemistry,

Department

The co~uersion

of androge~s

by a single F450

lyzed

conversion

the well-known requires stage

studies

the 19-aldehyde aldehydic

along

into estrogen

The other

ouriations radical

on the species.

two reactions,

oxidation theme,

~lec~anisti~ sequence,

oj’these

the qf&ementioned a~ternat~ue‘s

it anlike!\

data,

(tithe

alcohol

reactions,

step

The j&al

oxygen

in which

+

estrogen

mrchanism

were intro-

H20

bias

is used to

ure ~~lilniilated.

for

serue us

the hydroxylution

bond cleavage,

are viewed

courses for the neutralization

it is mt

oj The

is incorporated

atoms

f/3-> .?/3-, or (OP-h~droxy.~ter~)id,~

and C-IO-C-19

althorrgh

oxygen group.

of the 19_t,.~[\-orml~rostenPciionP

--;r -CH(OH)2

ulternative

also

reaction

route.

the process,

mechanisms

the conversion

are ~~~~7.~i~ier~~d and oar

-CHO,

as the hydroxyl

of

is the

of C-19 as ~~jr~~~~te. Our

process,

a fret> rndical

’ ’ representing

into

hydroxxlation

or via another

step

of the

that

We consider

‘ ‘hydroxylation For

used in the third

uia the se~l~t~nst~ -CHO

biosynthesis.

-CH@H

have showr? that the curhonyl

in the Jrst

at each stage

fn light

makes

the

~~,i~~~hare rata-

in the process

+ 02, thus representing

a second

to the aldehyde),

or those in which

analysi.~

rea~tion~~

NADPH

through

atid “0

UK

step

~l~~rnir~at~ng in the release

2H, “H, 02,

that

requires

converting

either

was introduced

numbers.”

is ~on~~idered to occur in estrogen

stage,

+ 0~.

from

bond cleuuugr

our mechanistic

intermediutes process.

that

d~st~~~ct generic

which

dehydrutes

It M’as jbund

as “whole

group

~~)ntuini~g

with another,

the C-IO-C-19

tn addition,

(which

Southampton,

1. The Jirst or P450~or,,mitru~i.i

The next

uses NADPH

is the one

formate.

or transferred

~f~volves three

he rationulized

using prerarsors

oxygen

promote

may

CH(OH)2 again

group

the released

duced

process.

02 und

a gem-dial,

in the process

extensive

into

+

estrogen ~art~matase

into a hydroxymethyl

hydroxylation

NADPH

producing

into

enzyme

of 19-methyl

ofSouthampton,

University

possible

is indi~~~ted.

to propose f Steroids

as

of

a trniqrte

%:I@-15

1.

1990)

Keywords:

steroids; aromatase: free-radical mechanism; P450: C-19 demethylation;

By the mid-1970s, the basic outline of the mechanism by which androgens are converted to estrogens was known.’ The conversion was viewed to involve three sequential oxidations (Figure iI, each requiring O2 and the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), which result in the 19-methyl group being first hydroxylated, then converted to the aldehyde, and, finally, excised as formic acid.2 Since it had previously been shown that during the overall process, l/3 and 2p hydrogen atoms of androgen are removed,3.4 it was assumed that the elimination of formate and the removal of the two hydrogen atoms are somehow mechanistically linked to the aromatization of the A-ring.

Address reprint requests to Dr. Muhammad Akhtar, Department of Biochemists. University of Southampton, Bassett Crescent East, Southampton, SO9 3TU UK.

142

Steroids,

1990, vol. 55, April

14wdemethylation:

sterols

The conversion of the IF)-alcohol 2 to the IPaidehyde 4 required O2 and NADPH rather than the NADP+ or NAD+ expected for a dehydrogenase-type mechanism. This unusual cofactor requirement was rationalized as a second hydroxylation giving rise to a gem-dial intermediate 3.5 The next question to be addressed was whetter the loss of 19-hydrogen in this reaction was stereospecific. This step was the first of two problems resolved in our laboratory by the use of stable isotopes. The second problem, dealt with in the second half of this article, was the origin and fate of the oxygen atoms involved as the C-19 methyl group is oxidized and cleaved from the steroid nucleus as formate.

Stereospecificity of oxidation at C-19 in estrogen biosynthesis The investigation of the stereochemistry of the oxidation converting the I9-alcohol 2 to the 19-aldehyde 4 required the synthesis of 1Phydroxy compounds la-

0 1990 Butterworth

Publishers

Isotope labels in aromatization

NADPH

studies:

Wright and Akhtar

+ H@

Q*

NADPH

+@

$2

r-t4 Homz.H

Pf”, J .

n

+ Hi.

-i

NADPH l2

+@ rj+

H’DH

lH,D

(4) (5)

Figure 1 The pathway for estrogen biosynthesis. The status of oxygen and hydrogen atoms during biosynthesis the gem-diol 3 is further considered in Figures 10 and 11. See Table 1 for key to symbols.

beled with tritium at the 19-HR, and 19-Hsi positions. It had already been observed that the reaction of methyl lithium with the 19-aldehyde in the Asv6series (6a, R = H or CH$O; Figure 2) gave rise to only one major isomer.6 This implied that other nucleophiles would also react with similar stereospecificity. Following this reasoning, two 194ritiated alcohols were prepared (Figure 2), one from the direct reduction of the 19aldehyde with NaB3H4 and the other from the reduction of [19-3H] aldehyde 6b with unlabeled NaBH4. These two alcohols, 7a and 7b (R = H), were incubated with human placental microsomes* in the presence of NADPH and 02, and the proportion of tritium liberated as 3Hz0 or 3HC02H was measured. With the alcohol 7a (R = H), the tritium was found predominantly in formate and, with 7b (R = H), the tritium was mainly in water. This demonstrated that the two alcohols, 7a and 7h, were labeled with a reasonable degree of stereospecificity and that the enzymic reaction could distinguish between the two pro-chiral hydrogens at C-19. The problem was then to assign an absolute stereochemistry to the two labeled compounds. A tentative assignment was made by analogy with the then-predicted steric course of reaction between methyl lithium and the 19-aldehyde? however, unambiguous information on this aspect became possible when, in collaboration with Professor Arigoni’s group

is shown. The role of

at ETH Zurich, a more rigorous method for the assignment of stereochemistry to the two C-19 hydrogen atoms of compounds of type 7 was developed.’ The basis of the assignment depicted in Figure 3 is as follows. When the 19-alcohol9 (R = H, 2H = H) is converted to the 19-acetoxy derivative 9 (R = CH&O, *H = H), the nuclear magnetic resonance (NMR) spectrum shows signals, from the C-19 diastereotopic hydrogens, as two doublets with distinct chemical shifts. After re-

(64

I

W)

NaB3H,

_&

I NaBH,

/I;&

Va)

Vb)

Figure 2 * The conversion of the 3/3-hydroxy-As%ystem into the conjugated ketone was achieved in situ by 3/3-hydroxysteroid dehydrogenasel isomerase present in placental microsomes.

The stereospecific introduction of hydrogen at C-19. That the 3H in 7a and 7b is predominantly in the Hsi and HRe orientation, respectively, is deduced from the approach in Figure 3.

Steroids,

1990, vol. 55, April

143

Papers

(8)

(9)

(10)

Figure 3 Arigoni’s method for the determination of the orientation of ZH introduced at C-19 from LiAIZH4. The alcohol 9 (R = HI was converted to 10 via the methanesulfonyl derivative 9 (R = CH3S02)

duction of the IPaIdehyde with LiAi2H4, the NMR spectrum of the acetoxy derivative 9 (R = CH30) showed a reduction in the intensity of the low field set to the reaction that ocof signals- corresponding curred with 80% stereospecificity (a slightly lower stereospecificity was observed for NaB2H4 reduction). To assign the absolute stereochemistry to the “low field” protons, the alcohol 9 (R = H) was converted to the 19-mesyl derivative 9 (R = CH3SOI) which, after solvolysis, formed the cyclopropane 10.As the chemical shifts of the two protons in the cyclopropane 10 were already known, it was possible to show that the deuterium was predominantly in the HRr position. On the well-supported assumption that the cyclopropane ring had been formed from the mesyl derivative by SN2 displacement, it was inferred that the precursor deutero-methoxy aicohol 9 (R = H) must have contained the deuterium in the Hfj position. Therefore, in the NMR spectrum, the “low field” doublet corresponds to the signals from the Hsi hydrogen. To return to the biologic experiment, it was now apparent that the [19-‘H] alcohols 6a and 6b and, hence, their Aring-conjugated ketone derivatives (structures of type 2) had the tritium in the Hv and HRu positions, respectively, and that it is the Hsi hydrogen that is retained during the enzymic oxidation while the HRr hydrogen is lost to water (Figure 1). This stereochemical conclusion was opposite that originally tentatively indicated on the basis of analogy with the methyl lithium reaction.2 The stereochemistry of the methyl lithium adduct has since been reassigned,R and the stereochemical course of the aromatase catalyzed conversion 2 -+ 4 has been confirmed by two independent approaches.*,”

Studies of the removal of C-19 in estrogen biosynthesis using l*O In 1974, Thompson and Siiteri’O showed that the overall stoichiomery of estrogen biosynthesis required three molecules of 02 and NADPH for each molecule of estrogen formed from an androgen precursor. This supported our earlier conclusion2 that the aromatization process occurs in three steps, each requiring 1 mol of O2 and 1 mol of NADPH. Evidence was also accumulating that an enzyme of the cytochrome P450 class was invoIved.r1~J2 The first two steps (Figure 1) could easily be related to normal cytochrome P450144

Steroids,

1990, vol. 55, April

catalyzed hydroxylation reactions, but the third step, while still requiring the same cofactors, was entirely novel. To gain further insight into this reaction, we performed a series of experiments using 180.‘3-‘5 In one set of experiments, 19-hydroxyandrostenedione 2 or 19-oxoandrostenedione 4 was incubated with human placental microsomes, containing NADPH, under r8O2 gas. The f o rmic acid released was isolated, converted to the benzyl ester, and examined for incorporation of I80 by mass spectral analysis. The early results indicated that a significant amount of label from r80,‘. gas was incorp orated into formate during the conversion of both the 19-hydroxy and 19-0~0 intermediates into estrogen. t3 In a complementary series of experiments [ 19-‘gO]hydroxyandrostenedione or [ 19-rgO]oxoandrostenedione were incubated with the enzyme under ‘“02 gas and the formate produced was similarly analyzed. i4,tS These experiments showed a retention of the r*O label in the formate released, from C-19 of the two intermediates. This latter experiment proved that the C-19 carbonyl group of 4 neither participates in Schiff-base formation nor is transferred to a carrier such as tetrahydrofolate during the cleavage reaction. At this time, several mechanisms for the hnai oxidation step had been proposed in the literature. They included l/3- or 2/3-hydroxylation3J6 (suggested because of the stereospecific loss of hydrogens from these positions during aromatization of the A-ring) and 4,5-epoxidation.” These proposals required a molecule of oxygen from water to be inco~orated into formate as part of the C-19-C-10 cleavage reaction (Figure 4). The I80 experiments described above showed that neither of the two oxygen atoms in formate could have come from water; thus, these types of mechanisms were (Figure 4) immediately ruled out. However, the oxygen atoms of the I/3- or 2@hydroxyi groups could become incorporated into formate if they formed a cyclic hemi-acetal with the IPaldehyde prior to cleavage of the C-IO-C-19 bond (Figure 5). The I@-hydroxylation mechanism was considered unlikely because of the unfavorable energetics involved in the formation of a four-membered ring, but the 2~-hydroxylation mechanism remains a viable possibility and will be further discussed later in this review. The 4,5-epoxy mechanism was also subsequently modified to allow incorporation of the epoxide oxygen into formate,rsa but no real evidence for this inte~ediate has been presented. As an alternative to

isotope labels in aromatization

-HCOOH -HO Mechanism A /

NADPH+O,

(4)

Mechanism B

-HCOOH

+P

0

OH

Figure 4 The cleavage of C-10-C-19 bond by mechanisms in which one of the oxygen atoms of formate is derived from H20.

hydroxylation on the A-ring, we favored a third oxidation at C-19, perhaps by nucleophilic attack of an enzyme-bound peroxy species, on the 19aldehyde to give 11 (Figure 6). The C-lo-C- 19 bond cleavage and the incorporation of oxygen into formic acid may then occur either directly through a cyclic mechanism (path a, Figure 6) or in a two-step process involving a lo@formate 12 (path b, Figure 6). The latter process has been eliminated by the demonstration that tritiated counterpart of this lO@formate 12 is not converted into estrogen by human placental microsomes.is Support for the fact that a peroxy complex of the type 11 may undergo C-C bond fission has recently come from a biomimetic experiment, lgbin which it was shown that the aldehyde 13 is slowly converted to the corresponding estrogen and formic acid in the presence of Hz02

studies:

Wright and Akhtar

(Figure 7). It was suggested that this conversion occurs through the involvement of a 19-peroxy species. Before we had completed our experiments, our attention was drawn to a proposal by Osawa and Shibatal which also involved a third oxygenation at C-19 and was also based on preliminary **Olabeling studies. This mechanism envisaged a third hydroxylation on the C-19 gem-diol, cleaving the C-10-C-19 bond in a concerted fashion and releasing orthoformate, which would immediately dehydrate to give formate. This mechanism required that only 33% of the oxygen used in the last step is incorporated into formate. In contrast, our mechanism (Figure 6) predicted that 100% of the oxygen used in the last step would be incorporated. To distinguish between these two mechanisms and to throw some light on the question of whether the aldehyde or its hydrate was the true intermediate, we needed to improve our technique for isolating and analyzing the labeled formate. The level of igO incorporation (50%) into formate found in our original experiments, l3 although higher than was predicted (33%) by the orthoformate mechanism, was not sufficiently beyond the limit of experimental error to warrant the elimination of the orthoformate mechanism. An improved methodology was, therefore, developedi5 in which steps were taken to minimize the exchange of I80 from substrates (i.e., the 19-aldehyde 4) and the product (formate) during the incubation. These included selecting a more suitable buffer to avoid exchange and reducing the incubation times. Extensive work was also done to find conditions for isolation and derivatization of formate which would allow this operation to be performed without disturbing its two oxygen atoms. It was found to be crucial to reduce the exposure of labeled formate to acid conditions as much as possible. Despite these precautions, the l*O content of the isolated formate was not as high or as repeatable as we would have liked. We suspected that the problem lay in the dilution of the biosynthetic for-

-HCOOH fi 0

HOU

Mechanism A /

I -HCOOH I

Mechanism B HO

Figure 5 The cleavage of C-10-C-19 a hemiacetal.

CHO

bond by mechanisms

OCHO

0-CHOH

in which oxygen introduced at either C-l or C-2 is transferred

Steroids,

to formate via

1990, vol. 55, April

145

Papers

HO

NADPH

+ 0,

t

Enz

(41

A

E nz-FEO-OH

h

61)

(4

Figure 6 Mechanistic alternatives showing the incorporation of oxygen from 0, into formate using a peroxide intermediate. uses an electrocyclic cleavage mode, whereas path b, which has been eliminated, I5 involves a Baeyer-Villiger rearrangement.

mate with the unlabeled material present in the enzyme preparation and the formation during the derivatization process of trace amounts of a compound which gave the same molecular ion as the unlabeled benzyl formate-these two factors contributing to an underestimation of the amount of IsO in formate. In order to bypass the contribution of these factors, the 19-alcohol 2 and the 19-aldehyde 4 were labeled with deuterium at C-19. (In the case of the 19-alcohol, this requires the deuterium to be in the Ha position because the HRe hydrogen is lost to water during the alcohol to aldehyde conversion.) By this method,r5 we were able to focus on the biosynthetic formate in the mass spectrometer because it (*HCOOH) was now one mass unit, and the corresponding igO species (*HCi800H) three mass units, heavier than the contaminants. This finally enabled us to get the high incorporations seen in Table 1.

Path a

tively, the results showed that the oxygen introduced into the 19-position in the first hydroxylation step as well as that used in the final cleavage reaction is quantitatively incorporated into formate (Figure 1). The oxygen used in the second oxidation, which converts the 19-alcohol 2 to the 19-aldehyde 4, is not found in any organic compound. If this oxidation does proceed via a gem-diol 3, it must then be stereospecifically dehydrated by the enzyme. Recently, a revised version of the orthoformate mechanism (Figure 8) has been pro-

Mechanistic analysis Mechanisms orthoformate.

operating

via

the

intermediacy

of an

The retention of 82% of the aldehydic oxygen and the incorporation of 90% of the label from oxygen gas in the last step 4 + 5 meant that we could eliminate any mechanism requiring less than quantitative retention/incorporation at this step. Cumula-

H8, Oestrogen

Figure 7 A non-enzymic cleavage of the C-IO-C-19 bond by a peroxide intermediate. The conversion was performed using H202/NaHC03. RI, t-butyldimethylsilyl; R2, tetrahydropyranyl.

146

Steroids,

1990, vol. 55, April

+ HCO@H

,OH

+ H,8

Figure 8 The cleavage of C-IO-C-19 bond using a gem-diol intermediate. The mechanism envisages a stereospecific loss of one of the three hydroxyl groups of CH(OHj3. See Table 1 for key to symbols.

isotope labels in aromatization Table 1

I80 Content of the formate

released from C-19 during estrone biosynthesis

studies:

using deuterated

Wright and Akhtar

substrates

Relative intensities of peaks due to benzyl ester of la0 atom % excess in *HCOzH *HC’*OOH 2HC180’80H substrate Gas phase (m/z 137) (m/z 139) (m/z 141)

Substrate Single ‘*O-labeling a 19-Oxo-(19-*t-l) (4, HA = *H) b IS-Oxo-(19-*H) (4, HA = *H) c 19-[‘B0]0xo-(19-2H,) (4, HA = *H, 0 = 180) d (IS.?+19-Hydroxy-(19-*Hl) (2, HA = *H) e (lSS)-IS-Hydroxy-(IS-*Hl) (2, HA = 2H) f (19S)-19-[‘60]Hydroxy-(19-2H’) (2, HA = *H, 0 = 160)

61 62

‘602 ‘no* (99%) ‘602 ‘602 ‘802 (99%) ‘602

99.5 5.4 47.0 99.5 6.6 41.9

0.25 90.0 49.8 0.25 86.5 56.1

0.25 4.6 2.2 0.25 7.9 2.0

Retention (ret) or incorporation (inc) of ‘*O in formate (%)

90 82 87 90

(inc) (ret) (inc) (ret)

Note. Variously labeled 19-oxo-4-androstene-3,17-dione and IS-hydroxy-4-androstene-3,17-dione were incubated with placental microsomes under atmospheres of 1602 or ‘sO2 as described in the text. The C-19 demethylation product, [*HIformic acid, was isolated and converted into benzyl formate in which the 180 content was measured by gas chromatography/mass spectrometry. Bold numerals refer to the structures in Figure 1.

posed in which the orthoformate fragment is cleaved and held at the active site, where it participates in the final enolization process (2P-hydrogen abstraction) before being stereospecifically dehydrated and released.20 We feel that this mechanism is improbable for two reasons. First, it is unlikely that the enzyme would be able to exert the required degree of stereochemical control over such a small fragment after it has been detached from the steroid nucleus. Second, using the [19-*80]aldehyde 4 (0 = ‘*O), we have noted a high retention of the labeled oxygen into formate (Table 1). If this reaction occurs by the above mechanism, then stringent stereochemical control would need to be exercised during two stages; a sterospecific hydration of the aldehyde to give the gem-dio13, then a stereospecifit dehydration of the orthoformate. Such a scenario is conceivable, but only remotely. We think our I80 labeling results are more consistent with the I9-aldehyde (rather than the gem-diol) being the substrate for the final oxidation. This left our suggestion of a peroxytype mechanism as one of the few remaining alternatives. With the purification of the estrogen biosynthetic enzyme system,21-23 it became clear that it was indeed a cytochrome P450 (now designated cytochrome P45t&Xn.&,,,, and that a single enzyme was responsible for catalyzing all three steps of the reaction, as had

NADPH+O, CH, I

R

HOW,

( Fe”’ )

+

-

-OH [ Fe”‘-0OH (34)

P-450 Aromatase

(

[F$OH:HZ]

CH,

] /

1 R

[ Fe”=0 (‘5)

CH,

]

1 R

-

Figure 9 A free radical mechanism for the P450-dependent droxylation process. R, steroid nucleus.

hy-

been predicted earlier. At the same time, a reexamination of the stereochemistry of cytochrome P450-catalyzed hydroxylation reactions led to the conclusion that they proceed via stepwise radical processes24-26 rather than the previously favored concerted mechanisms.27-29 These new insights have enabled us to rationalize the three aromatase reactions as occurring by variations of related radical processes,30 which are considered below.

A radical mechanism for the various reactions catalyzed by aromatase Hydroxylation at C-19. It is now widely thought that cytochrome P450 catalyzed hydroxylations at unactivated carbon atoms proceed via abstraction of a hydrogen by an iron-monooxygen species such as the ferroxy radical represented by the canonical structure Feiv-0 t, Fe’ = 0 (14, Figure 9). This leads to the formation of a carbon radical that is neutralized by association with a hydroxyl radical (Figure 9). This is often referred to as the oxygen rebound mechanism. Oxidation of lkalcohol(2) to lkaldehyde (4). Conversion of the 19-alcohol 2 to the 19-aldehyde 4 may be rationalized by either of the two processes shown in Figure 10. After initial abstraction of the HRe (as described above for the first hydroxylation), the reaction could then proceed either by (1) normal oxygen rebound to give the gem-diol followed by stereospecific dehydration (path a) or (2) a hydrogen abstraction from the hydroxyl group, producing the carbonyl group (path b). The retention of the hydroxyl oxygen atom in the aldehyde group shown by our la0 experiments would be consistent with the more direct path b. Recent experiments, 2o however, have shown the incorporation of I80 from 1802 gas into a C-19 carbonyl during the oxidation of an analogue (16) of the 19alcohol by aromatase (Figure 11). In this case, a gemdiol is presumably formed, but this does not exclude path b for the true substrate. Steroids,

1990, vol. 55, April

147

Papers

HO;