Mechanistic consequences of chiral radical clock probes: Analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates

Article abstract of DOI:10.1021/ja4100035

(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.

Full text of DOI:10.1021/ja4100035

Communication
pubs.acs.org/JACS
Mechanistic Consequences of Chiral Radical Clock Probes: Analysis of
the Mononuclear Non-Heme Iron Enzyme HppE with 2‑Hydroxy-3-
methylenecyclopropyl Radical Clock Substrates
Hui Huang,†,‡ Wei-Chen Chang,†,‡ Geng-Min Lin,† Anthony Romo,† Pei-Jing Pai,§ William K. Russell,§
David H. Russell,§ and Hung-Wen Liu*,†
†Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, University of Texas at Austin, Austin, Texas
78712, United States
§Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
S
* Supporting Information
number of studies provide evidence for the radical intermediates
during turnover.3
ABSTRACT: (S)-2-Hydroxypropylphosphonic acid [(S)-
HPP] epoxidase (HppE) is a mononuclear iron enzyme
that catalyzes the last step in the biosynthesis of the
antibiotic fosfomycin. HppE also processes the (R)-
enantiomer of HPP but converts it to 2-oxo-propylphos-
phonic acid. In this study, all four stereoisomers of 3-
methylenecyclopropyl-containing substrate analogues, (2R,
3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were
synthesized and used as radical probes to investigate the
mechanism of the HppE-catalyzed reaction. Upon treat-
ment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were
converted via a C1 radical intermediate to the correspond-
ing epoxide products, as anticipated. In contrast,
incubation of HppE with (2R, 3R)-8 led to enzyme
inactivation, and incubation of HppE with (2R, 3S)-8
yielded the 2-keto product. The former finding is
consistent with the formation of a C2 radical intermediate,
where the inactivation is likely triggered by radical-induced
ring cleavage of the methylenecyclopropyl group. Reaction
with (2R, 3S)-8 is predicted to also proceed via a C2
radical intermediate, but no enzyme inactivation and no
ring-opened product were detected. These results strongly
suggest that an internal electron transfer to the iron center
subsequent to C−H homolysis competes with ring-
opening in the processing of the C2 radical intermediate.
The different outcomes of the reactions with (2R, 3R)-8
and (2R, 3S)-8 demonstrate the need to carefully consider
the chirality of substituted cyclopropyl groups as radical
reporting groups in studies of enzymatic mechanisms.
(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE), a
mononuclear non-heme iron enzyme, performs an epoxidation
reaction on (S)-2-hydroxypropylphosphonic acid ((S)-HPP, 1)
to form fosfomycin (2) in the last step of the biosynthesis of this
clinically useful antibiotic.4,5 Previous studies showed that the
epoxide ring of 2 is generated by an initial abstraction of 1-HR
from (S)-HPP and subsequent C−O bond formation between
C1 and the 2-OH group of (S)-HPP in a dehydrogenation−
cyclization manner (Scheme 1A).4−7 When HppE is incubated
with (R)-HPP (3), the 2-H is selectively abstracted, leading to
direct oxidation of the alcohol moiety to give 2-oxopropylphos-
phonic acid (4) as the sole product (Scheme 1B).8 Crystallo-
graphic and spectroscopic experiments have shown that both
Scheme 1. Proposed Mechanism for the Oxidation of (S)- and
a
(R)-HPP by HppE
ononuclear non-heme iron enzymes are an important
1
Mclass of biocatalysts that are widespread in nature. The
catalytic functions of these enzymes are remarkably diverse, but
the involvement of a high valent Fe(IV)-oxo (ferryl) species as a
reactive intermediate is common to all their catalytic
mechanisms. This reactive iron−oxygen species abstracts a
hydrogen atom, typically from an unactivated carbon of the
substrate, to generate a substrate radical, which then undergoes
further transformations en route to the final product.2 Despite
the proposed intermediacy of substrate radicals in various
mononuclear non-heme iron enzyme mechanisms, only a limited
a
(A) Conversion of (S)-HPP (1) to fosfomycin (2). (B) Conversion
of (R)-HPP (3) to 2-oxopropylphosphonic acid (4). (C) Conversion
of (S)-7 to 9 and (R)-7 to 10 by HppE. (D) Proposed mechanisms for
the inactivation of HppE by 8.
Received: October 2, 2013
Published: February 10, 2014
© 2014 American Chemical Society
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(S)- and (R)-HPP bind to the iron center of the enzyme as
bidentate ligands.9 The C2 chirality of the substrates governs the
binding geometry such that regio- and stereoselective hydrogen
atom abstraction (1-HR for (S)-HPP and 2-H for (R)-HPP) is
facilitated by the reactive iron−oxygen species10,11 giving
different radical intermediates (5 or 6).12
limit the free rotation of the C2−C3 bond connecting the two
neighboring chiral centers and, therefore, magnify the diastero-
meric difference. This strategy generated two TBDPS-ethers,
(2S, 3R)- and (2R, 3R)-16, which were successfully separated by
silica gel chromatography. Removal of the TBDPS group yielded
pure (2S, 3R)- and (2R, 3R)-15, enabling chirality and
enantiomeric excess determination of the C2 alcohol by the
Mosher ester method.15 Subsequent deprotection by TMSBr
gave the desired substrate analogues (2S, 3R)- and (2R, 3R)-8.
Following the same procedure, (2S, 3S)- and (2R, 3S)-8 were
obtained using (S)-13 as the starting material.
Recently, we reported mechanistic studies of HppE with the 2-
hydroxyl-3-cyclopropyl and 2-hydroxyl-3-methylenecyclopropyl
compounds (S)-7, (R)-7, and (rac)-8.13 Both (S)- and (R)-7
were efficiently converted by HppE to the epoxide and ketone
products 9 and 10, respectively (Scheme 1C). In contrast, when
HppE was incubated with (rac)-8, only a small portion of the
substrate was converted to new products before the enzyme was
inactivated. It was suggested that inactivation results from ring-
opening of the C3 methylenecyclopropyl group triggered by the
C2 radical (11) generated during turnover (Scheme 1D). These
studies provided preliminary evidence for a radical intermediate
in the HppE-catalyzed reactions.13 However, (rac)-8 used in the
experiment was a mixture of four stereoisomers, which
complicated the analysis of the reaction outcomes and thus
hampered investigation of the inactivation mechanism. To gain
further mechanistic insight and verify our previous observations,
we synthesized all four stereoisomers of 8 and studied their
reactions with HppE. Our results, reported herein, show that
HppE reactivity toward different diasteromers of 8 is dictated not
only by the chirality of the C2 hydroxyl group but also the
methylenecyclopropyl substituent at C3. Specifically, it was
found that incubation of the (2R, 3R)-isomer with HppE leads to
enzyme inactivation, whereas reaction with the (2R, 3S)-isomer
gives rise to complete turnover to a 2-keto product. These results
are mechanistically informative and shed light on the likely
formation of a carbocation intermediate in HppE catalysis.
The syntheses of all four diastereomers of 8 began by
addressing the chirality of the C3 methylenecyclopropyl group.
This was achieved by following the previously reported
enantioselective synthetic method for alcohols (S)- and (R)-
13.14 Oxidation of (R)-13 produced aldehyde (R)-14, and
subsequent nucleophilic addition with methyldiethylphospho-
nate gave (2S, 3R)- and (2R, 3R)-15 as a pair of diastereomeric
alcohols (Scheme 2). Direct separation or enzymatic resolution
The four purified diastereomers of 8 were each incubated with
1
HppE, and the reactions were monitored using real-time H
NMR spectroscopy. All four compounds were substrates for
HppE, but each reaction gave a different result. The 1,2-epoxide
17 was produced when (2S, 3S)-8 was employed as the substrate,
whereas formation of a different 1,2-epoxide, 18, was detected in
the reaction with (2S, 3R)-8 (see Figures S3, S4).16 These results
are consistent with what was previously observed for (rac)-8 with
HppE13 and support the regio- and stereoselective conversions of
(2S, 3S)- and (2S, 3R)-8 proposed in Scheme 1A. Specifically,
when these substrate analogues bearing C2-(S) hydroxyl group
react with HppE, the putative C1 centered radicals (19 and 20)
are produced by selective hydrogen abstraction with the high-
valent Fe(IV)-oxo species. These C1 radicals are not affected by
the methylenecyclopropyl group at C3 position and undergo
cyclization to the corresponding epoxides 17 and 18 in a manner
similar to the formation of 2 from 1 (Scheme 3). Our more
Scheme 3. Conversion of (2S, 3S)-8 to 17 and (2S, 3R)-8 to 18
by HppE
Scheme 2. Synthetic Scheme for the Preparation of (2S, 3R)-
and (2R, 3R)-8
recent results suggest that cyclization of the resulting radical
intermediate (19 and 20) to the epoxide product likely involves a
carbocation intermediate (21 and 22) via internal electron
transfer to reduce the iron center.17
Based on the mechanism proposed in Scheme 1B, incubation
of the C2-(R) analogue, (2R, 3R)-8, with HppE is expected to
proceed with regioselective C-2 hydrogen atom abstraction,
where the resulting C2 radical will trigger ring-opening of the
neighboring methylenecyclopropyl group18 leading to enzyme
inactivation. Interestingly, incubation of (2R, 3R)-8 with HppE
produces a small amount of new product as detected by 1H NMR
spectroscopy (see Figure S1). However, product formation
stopped with no further consumption of the substrate after ∼10
min. Moreover, no fosfomycin could be detected when the
natural substrate, (S)-HPP (1), was incubated with HppE that
had been preincubated with (2R, 3R)-8, suggesting complete
(using lipases or esterases) of these two diastereomers did not
give satisfactory results. Consequently, we focused on chemical
resolution by derivatizing the C2 hydroxyl group with a TBDPS
group to inflict internal steric hindrance and to introduce a UV
chromophore for product detection during purification. It was
hoped that the attachment of such a bulky substituent would
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Communication
inactivation of HppE by the pretreatment. The turnover product
of this process was later determined to be ketone 23, which had
been previously shown not to be an inactivator of HppE.13 Thus,
a logical explanation for the inactivation of HppE by (2R, 3R)-8 is
a mechanism initiated by a radical-triggered ring-opening of the
C3-(R)-methylenecyclopropyl group (Scheme 4A). The fact that
different amounts of (2R, 3R)-8, a plot of the residual enzyme
activity versus the equivalents of (2R, 3R)-8 employed resulted in
a partition ratio of 0.5 (Figure S6), reflecting that ∼1.5 equiv of
(2R, 3R)-8 are required to fully inactivate 1.0 equiv of HppE.
Moreover, the inactivation appears to be irreversible since
enzyme activity could not be regenerated after extensive dialysis.
Tritiated (rac)-[3-3H]-8 was also synthesized and incubated
with HppE to determine the stoichiometry of inactivation.16
After HppE was completely inactivated by (rac)-[3-3H]-8, the
reaction mixture was subjected to size exclusion chromatography
followed by dialysis to remove unbound small molecules. About
1.1 equiv of tritiated compound per enzyme were found to be
associated with the inactivated HppE.16 Interestingly, when this
protein sample was denatured by boiling, over 80% of the
radioactivity was lost from the protein fraction after size
exclusion centrifugal filtration.16 In addition, no covalently
modified peptide fragment was detected by mass spectrometry
analysis of the inactivated HppE after denaturation and tryptic
digestion (see Figure S7). Taken together, these results strongly
suggest that the inactivation is not due to covalent modification
of HppE, as previously proposed,13 but is likely due to tight
binding of a ring-opened product to the enzyme active site.
It is conceivable that the causative agent for the observed
inactivation is compound 29, which arises from canonical
hydroxyl group rebound to the initially formed acyclic radical
intermediate (12, Scheme 5). The tridentate coordination of 29
Scheme 4. (A) HppE Inactivation by (2R, 3R)-8 and (B)
Conversion of (2R, 3S)-8 to 26 by HppE
Scheme 5. Proposed HppE Inactivation by the Ring-Opened
Product Derived From (2R, 3R)-8
(2R, 3R)-8 is an inactivator and also a substrate for HppE reveals
a competition between the radical-induced ring-opening (24 →
12) and the internal electron-transfer (24 → 25) reactions.
For the other C2-(R) diastereomer, (2R, 3S)-8, a similar C2
radical-triggered ring-opening reaction with subsequent enzyme
inactivation was also expected. However, HppE remained active
after incubation with (2R, 3S)-8, and NMR analysis of the
incubation showed that (2R, 3S)-8 was completely transformed
into ketone 26 without formation of ring-opened products
(Scheme 4B and Figure S2). Given that the methylenecyclo-
propyl group is a highly sensitive radical probe,18,19 the different
outcomes of these two reactions may lie in the orientation of the
methylenecyclopropyl group of 8 in the enzyme active site.
Specifically, the three-membered ring is more readily opened
when the C3−C4 bond of the methylenecyclopropyl group and
the singly occupied orbital of the radical adopt an anticoplanar
conformation18 (the ring-opening rate of a methyenecyclopropyl
carbinyl radical in free solution is ∼6 × 109 s−1).19 It is possible
that such an anticoplanar conformation is inaccessible for (2R,
3S)-8 due to steric interference between the methylenecyclo-
propyl moiety and the groups at the enzyme active site. Thus,
electron transfer from the radical intermediate 27 to the iron
center (27 → 28) becomes the path of choice leading to a 2-keto
product (26) instead of the ring-opened product(s).
To gain more insight into the mechanism of inhibition by (2R,
3R)-8, the kinetics of the reaction were determined using HPLC.
When HppE was incubated with 10-fold excess of (2R, 3R)-8,
time-dependent inactivation was observed, and the enzyme
completely lost activity within 3 min (Figure S6). When (S)-HPP
(1) was coincubated with (2R, 3R)-8 (molar ratio of 1:8 = 20:1),
fosfomycin production was still discernible after 8 min, indicating
a reduction of the rate of inactivation in the presence of native
substrate. The observed substrate protection implies that the
inhibition is active-site directed. When HppE was treated with
to the iron center could render 29 to bind strongly in the active
site of HppE, resulting in enzyme inactivation. However, efforts
to isolate and identify this putative tight binding species after
protein denaturation were unsuccessful. It was later found during
our attempt to chemically synthesize 30 (the keto tautomer of
29) that this compound readily undergoes intramolecular
cyclization to give 32 as the main product.16 The fact that a
low abundance signal at m/z 175 [M − 1]− was detected by LC-
MS analysis in the denatured enzymatic sample (but not in the
control) is consistent with the presence of 32 in the protein-free
solution after enzyme denaturation. Unfortunately, the low
abundance of this signal prevented MS-MS analysis to further
verify its structure.
The results presented herein are significant for three reasons.
First, a highly effective inhibitor for HppE, (2R, 3R)-8, has been
identified. The inactivation is mechanism-based, since assays
using NMR, HPLC, MS, and radioactive methods indicate that
the inactivation arises from the methylenecyclopropyl ring-
opening triggered by the C2-centered radical. However, unlike
most known suicide inhibitors, the inactivation by (2R, 3R)-8
does not involve covalent modification of the targeted enzyme
but is likely due to tight binding of a ring-opened product in the
active site.
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Commun. 1987, 1661. (b) Ryle, M. J.; Liu, A.; Muthukumaran, R. B.; Ho,
R. Y. N.; Koehntop, K. D.; McCracken, J., Jr.; Que, L.; Hausinger, R. P.
Biochemistry 2003, 42, 1854. (c) Howard-Jones, A. R.; Elkins, J. M.;
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Second, to “capture” radical intermediate(s) in a reaction with
transient lifetimes, the sensitivity of the cyclopropylcarbinyl
radical probe can be fine-tuned by adding substituent(s) to the
cyclopropyl ring.18 Such modifications usually introduce new
chiral center(s) and inflict steric demands upon binding to the
chiral environment of the active site. However, the effect of the
chirality of the substituted cyclopropyl ring of the probe on the
outcomes of the radical triggered ring-opening reaction has rarely
been addressed.20,21 In this study, enzymatic reactions with (2R,
3R)- and (2R, 3S)-8 led to different results, even though both are
believed to involve a C2 radical intermediate. The distinct
outcomes may simply reflect different binding geometries of (2R,
3R)- and (2R, 3S)-8 in the enzyme active site, and underscores
the need to consider the chirality of substituted cyclopropyl
moiety in a radical probe and its binding mode in the active site in
studies of enzyme mechanisms. One also needs to be cautious to
interpret the experimental data when no predicated ring-
opening/rearrangement happens.
Third, the intermediacy of a substrate-derived cation in the
catalytic cycle of HppE was recently proposed based on the
observed 1,2-phosphono migration with an alternative substrate
(R)-1-HPP.17 The observation of both enzyme inactivation and
2-keto product formation upon incubation of HppE with (2R,
3S)-8 is mechanistically significant, as it reveals that an internal
electron transfer (leading to the 2-keto product 23) is a
competing pathway with ring-opening (leading to enzyme
inactivation) in the processing of the C2 radical intermediate.
Thus, these results provide additional evidence supporting the
formation of a cation intermediate in HppE catalysis and permit
estimation of the rate constant for internal electron transfer to be
on the order of ∼6 × 109 s−1, which is the reported rate constant
for ring-opening of the methylenecyclopropylcarbinyl radical.19
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ASSOCIATED CONTENT
* Supporting Information
Details of experimental conditions and procedures. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
■
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Author Contributions
‡These authors contributed equally.
(16) See SI for experimental details.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Steve Sorey for his help in acquiring the NMR spectra.
This work is supported in part by grants from the National
Institutes of Health (GM040541 to H.-W.L.) and the Welch
Foundation (F-1511 to H.-W.L. and A-1176 to D.H.R.).
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