Combining a Clostridial Enzyme Exhibiting Unusual Active Site Plasticity with a Remarkably Facile Sigmatropic Rearrangement: Rapid, Stereocontrolled Entry into Densely Functionalized Fluorinated Phosphonates for Chemical Biology

Article abstract of DOI:10.1021/jacs.5b00022

Described is an efficient stereocontrolled route into valuable, densely functionalized fluorinated phosphonates that takes advantage of (i) a Clostridial enzyme to set the absolute stereochemistry and (ii) a new [3,3]-sigmatropic rearrangement of the thiono-Claisen variety that is among the fastest sigmatropic rearrangements yet reported. Here, a pronounced rate enhancement is achieved by distal fluorination. This rearrangement is completely stereoretentive, parlaying the enzymatically established β-C-O stereochemistry in the substrate into the δ-C-S stereochemistry in the product. The final products are of interest to chemical biology, with a platform for Zn-aminopeptidase A inhibitors being constructed here. The enzyme, Clostridium acetobutylicum (CaADH), recently expressed by our group, reduces a spectrum of γ,δ-unsaturated β-keto-α,α-difluorophosphonate esters (93-99% ee; 10 examples). The resultant β-hydroxy-α,α-difluorophosphonates possess the L -stereochemistry, opposite to that previously observed for the CaADH-reduction of ω-keto carboxylate esters ( D ), indicating an unusual active site plasticity. For the thiono-Claisen rearrangement, a notable structure-reactivity relationship is observed. Measured rate constants vary by over 3 orders of magnitude, depending upon thiono-ester structure. Temperature-dependent kinetics reveal an unusually favorable entropy of activation (ΔS^? = 14.5 ± 0.6 e.u.). Most notably, a 400-fold rate enhancement is seen upon fluorination of the distal arene ring, arising from favorable enthalpic (ΔΔH^? = -2.3 kcal/mol) and entropic (ΔΔS^? = 4 e.u., i.e. 1.2 kcal/mol at rt) contributions. The unusual active site plasticity seen here is expected to drive structural biology studies on CaADH, while the exceptionally facile sigmatropic rearrangement is expected to drive computational studies to elucidate its underlying entropic and enthalpic basis. (Chemical Presented).

Full text of DOI:10.1021/jacs.5b00022

Article
pubs.acs.org/JACS
Combining a Clostridial Enzyme Exhibiting Unusual Active Site
Plasticity with a Remarkably Facile Sigmatropic Rearrangement:
Rapid, Stereocontrolled Entry into Densely Functionalized
Fluorinated Phosphonates for Chemical Biology
Kaushik Panigrahi,§ Gregory A. Applegate,§ Guillaume Malik, and David B. Berkowitz*
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, United States
S
* Supporting Information
ABSTRACT: Described is an efficient stereocontrolled route into
valuable, densely functionalized fluorinated phosphonates that
takes advantage of (i) a Clostridial enzyme to set the absolute
stereochemistry and (ii) a new [3,3]-sigmatropic rearrangement of
the thiono-Claisen variety that is among the fastest sigmatropic
rearrangements yet reported. Here, a pronounced rate enhance-
ment is achieved by distal fluorination. This rearrangement is
completely stereoretentive, parlaying the enzymatically established
β-C−O stereochemistry in the substrate into the δ-C−S
stereochemistry in the product. The final products are of interest
to chemical biology, with a platform for Zn-aminopeptidase A
inhibitors being constructed here. The enzyme, Clostridium
acetobutylicum (CaADH), recently expressed by our group, reduces
a spectrum of γ,δ-unsaturated β-keto-α,α-difluorophosphonate
esters (93−99% ee; 10 examples). The resultant β-hydroxy-α,α-difluorophosphonates possess the “L”-stereochemistry, opposite
to that previously observed for the CaADH-reduction of ω-keto carboxylate esters (“D”), indicating an unusual active site
plasticity. For the thiono-Claisen rearrangement, a notable structure−reactivity relationship is observed. Measured rate constants
vary by over 3 orders of magnitude, depending upon thiono-ester structure. Temperature-dependent kinetics reveal an unusually
favorable entropy of activation (ΔS‡ = 14.5 0.6 e.u.). Most notably, a 400-fold rate enhancement is seen upon fluorination of
the distal arene ring, arising from favorable enthalpic (ΔΔH‡ = −2.3 kcal/mol) and entropic (ΔΔS‡ = 4 e.u., i.e. 1.2 kcal/mol at
rt) contributions. The unusual active site plasticity seen here is expected to drive structural biology studies on CaADH, while the
exceptionally facile sigmatropic rearrangement is expected to drive computational studies to elucidate its underlying entropic and
enthalpic basis.
In developing the synthetically useful biocatalytic chemistry
that will form the basis of the retrosynthetic transform arrows3
that Turner’s commentary envisions, three broad approaches
may be pursued: (i) panning the biosphere for wild type
enzymes of utility in synthesis,4 (ii) engineering enzymes from
natural protein templates through directed evolution,5 or (iii)
de novo design of active sites with the aid of modern
computational techniques.6 Whichever approach is taken, the
optimal solution will lead to enzymes that not only are able to
set absolute stereochemistry in synthetically complex substrates
but also ideally will show significant substrate generality and, as
such, can be treated much as a “privileged” chiral organic or
organometallic catalyst would be.7
INTRODUCTION
■
In modern asymmetric synthesis and its translation into process
chemistry applications on scale, we have entered an age in
which the careful melding of sophisticated enzymatic chemistry
with sophisticated organic chemistry can present state-of-the-art
solutions. In thinking about this evolution, Turner has
suggested that we rethink how we analyze synthetic challenges
to include a dimension of “biocatalytic retrosynthesis.”1 In so
doing, it is important to move beyond the mindset that
enzymes are solely useful for the delivery of small molecule
building blocks, and to more frequently embrace the notion of
challenging enzymes with substrates of higher carbon count
and/or that are decorated with a more complex array of
functionality. One of the best examples of such a sophisticated
hybrid asymmetric synthesis is the current Merck process for
the synthesis of sitagliptin in which an engineered transaminase
sets the absolute stereochemistry in acting upon an advanced
16-carbon substrate bearing 12 heteroatoms.2
We describe herein such an approach that melds a new
stereoselective enzymatic transformation to set the absolute
stereochemistry for a C−O bond in a densely functionalized
Received: January 2, 2015
Published: February 26, 2015
© 2015 American Chemical Society
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fluorinated phosphonate environment, with a new type of
hetero-Claisen rearrangement that parlays the established β-C−
O bond stereochemistry into δ-C−S bond stereochemistry,
with stereochemical fidelity and remarkable facility, via formal
allylic transposition. The enzymatic transformation avails itself
of a Clostridial alcohol dehydrogenase (CaADH) recently
expressed and characterized by our group,8 but identifies a
previously unexplored substrate class for this enzyme, or any
dehydrogenase for that matter, namely β-keto-α,α-difluoro-
phosphonates, thereby generating a retrosynthetic transform
arrow not previously available in the biocatalytic toolbox.
Perhaps most importantly, these studies reveal a sort of “active
site plasticity”9 rarely seen, in which two distinct substrate
classes are each reduced with high enantioselectivity in the
same active site, but with opposite facial selectivity. Molecular
modeling is used to examine how these two substrate classes
bind with high facial bias for hydride transfer, but in quite
different three-dimensional arrangements in the CaADH active
site. The chemical step introduces a rearrangement (RR) of the
[3,3]-sigmatropic variety that rivals even the classic Evans−
Golob anion-accelerated oxy-Cope rearrangement in terms of
rate10 and that is worthy of note (i) for its unusually favorable
entropy of activation, (ii) for its stereoretentive nature, and (iii)
for the remarkable 400-fold gain in rate that is seen upon
fluorination of the flanking aromatic ring in the thionocar-
bonate system (vide infra).
As is illustrated in Figure 1, the melded process constitutes a
new retrosynthetic approach to the stereocontrolled synthesis
of δ-thio-α,α-difluorinated phosphonates that both possess an
intervening functionalizable alkene and that may be decorated
with additional functionality, as desired for chemical biological
purposes. In the case at hand, we provide proof of principle, by
utilizing this hybrid biocatalytic/pericyclic sequence to
construct a platform for potential zinc-aminopeptidase A (Zn-
APA) inhibitor development. Zn-APA converts angiotensin II
into angiotensin III in the brain and is an emerging target in
hypertension.11
Phosphonates12 bearing varying degrees of α-fluorination are
of considerable value to medicinal chemistry13 and chemical
biology.14 To be sure, we15,16 and others17 have had a
longstanding interest in synthetic routes into this compound
class, dating back to the early proposition by Blackburn18 and
McKenna,19 that α,α-difluoroalkyl phosphonates are “isopolar”
analogues of the corresponding phosphate esters. Importantly,
these “teflon phosphates” are inert to digestive phosphatase
enzymes and, therefore, serve as useful tools in chemical
biology. For example, we were the first to synthesize the CF2-
phosphonate analogues of phosphoserine and phosphothreo-
nine,16 and these have proven to be very useful chemical
mimics for the “constitutive phosphorylation” phenotype in
studying kinase regulation in the cell.20
The CaADH reduction/sigmatropic rearrangement sequence
here constitutes a new approach into densely functionalized,
α,α-difluorinated phosphonates, bearing β,γ-unsaturation and δ-
thio substitution. As is illustrated in Figure 1, this substitution
pattern maps onto thio-substituted peptide,21 β-lactam,22 and
phospholipid23 scaffolds of interest in chemical biology, as well
as thio-nucleic acid systems exploited by Piccirilli24 and
Szostak.25 In none of these systems are the corresponding
difluorinated phosphonates yet known, as all bear phosphate
esters or simple CH2−phosphonate functionalities δ to the
sulfur.
Figure 1. Stereocontrolled route into densely functionalized δ-thio-
α,α-difluorophosphonates of interest to chemical biology. (Top)
CaADH is challenged with a new ketophosphonate substrate class to
set the absolute stereochemistry, and a new [3,3]-sigmatropic RR is
employed to parlay the enzymatically established β-C−O stereo-
chemistry into the δ-C−S center. (Bottom) Mapping the δ-thio-α,α-
difluorophosphonate motif onto lipid, carbohydrate, and β-lactam
scaffolds of interest to chemical biology.
As alluded to above, to demonstrate proof of principle, the
focus here is on a platform related to the δ-thio-phosphonate
embedded within a β-amino acid motif developed by Roques21
that serves as the key residue in tripeptide inhibitors of Zn-
aminopeptidase A (APA; potential new target for antihyper-
tensives; Figure 1). Note that the thiol here is critical for Zn2+
coordination and that compound 3e, reported herein (vide
infra), represents a very useful platform upon which to build
APA inhibitor candidates, as it bears three of the four requisite
functional groups (phosphonate, thiol, and carboxylic acid) all
properly positioned, with α,α-difluorination. Thus, successful
synthetic entry into this platform sets the stage for future
studies directed at library generation, particularly through
functionalization of the intervening alkene.
To put the subsequent sigmatropic rearrangement step into
perspective, the Claisen rearrangement has been in the domain
of synthetic chemistry for over a century26 and is also in
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a
Nature’s repertoire of enzymatic transformations. Most notably,
the enzyme chorismate mutase catalyzes a Claisen rearrange-
ment as a key step in the biosynthesis of the aromatic amino
acids. Recently, the groups of Tanner27 and Schmidt28 have
discovered that Nature apparently exploits other [3,3]-
sigmatropic rearrangements, an aromatic Cope and Claisen
rearrangement, respectively, in formal biosynthetic prenylation
reactions, as well. In the domain of chemical biology as well,
great efforts have been expended to understand and mimic
chorismate mutase, including two of the earliest examples of
catalytic antibodies from Hilvert29 and Schultz,30 and
contemporary studies of protein dynamics.31
Scheme 1. Synthesis of β-Ketophosphonate Substrates
The nonenzymatic reaction generally proceeds at elevated
temperature and requires an organized transition state,
prompting considerable effort at developing synthetic catalysts,
including a supramolecular, tetrahedral M4L6 assembly32 and an
elegant spiroligozyme system.33 The former promotes an aza-
Claisen rearrangement by preorganizing an otherwise entropi-
cally unfavorable reaction leading to a small entropic driver for
the catalyzed process (ΔS‡uncat = −8 e.u.; ΔS‡cat = +2 e.u.). The
latter achieves a 58-fold acceleration of an aromatic Claisen
rearrangement, presumably via H-bonding catalysis.
a
(a) Aldehyde addition/DMP oxidation route; yields reflect isolated
yield following two-step procedure. (b) Direct ester addition route; all
yields are for isolated pure compounds.
There are precedents for what are formally thermal and
metal-catalyzed [3,3]-sigmatropic rearrangements of the
thiono-Claisen variety (vide infra). However, these trans-
formations generally require elevated temperatures and
significant reaction times. For example, thionocarbonate
rearrangements reported by Crich and co-workers required
refluxing in benzene for 4 h.34 There are also early reports from
Nakai35 and Zaim36 about simple thionocarbonate and thiono
chloroformate-type rearrangements, respectively, though these
communications are accompanied by very limited experimental
detail. The former gives a measured kobs corresponding to a
half-life of over 104 min at 80 °C for a thionocarbamate
rearrangement and claims that the corresponding xanthate is
significantly faster (data not given; trend is opposite to the data
reported herein). The latter reports on very reactive
chloroformates, generated in situ from thiophosgene, that are
unfunctionalized, difficult to handle, and of limited synthetic
utility. Stereochemical control does not enter into these studies.
A single kobs is reported, corresponding to a half-life of 60 min
at rt.
nated β-ketophosphonates bearing β,γ-unsaturation (Note: E/Z
ratio >20:1 in all cases) in good yield and with excellent
enantioselectivity. The results are displayed in Scheme 2. The
expensive nicotinamide cofactor was regenerated with glucose
dehydrogenase, allowing D-glucose to serve as the stoichio-
metric carbonyl reductant.
a
Scheme 2. Substrate Scope-CaADH-Mediated Reductions
RESULTS AND DISCUSSION
■
α,α-Difluoro-β-keto-phosphonate Synthesis and
Asymmetric CaADH Reduction. As is illustrated in Scheme
1, the requisite keto phosphonates were prepared by
condensation of LiCF2P(O)(OEt)2 with methyl cinnamate
and related β-aryl-α,β-unsaturated esters. A two-step procedure
proved to be more efficient for systems bearing aliphatic
substituents at the β-position. In such cases, the β-aryl-α,β-
unsaturated aldehyde was condensed with LiCF2P(O)(OEt)2,
followed by oxidation with the Dess-Martin periodinane.
The Berkowitz laboratory has a longstanding interest in the
creative use of enzymes in asymmetric synthesis,8,37 and
recently we described an alcohol dehydrogenase from
Clostridium acetobutylicum (CaADH) that is particularly
effective for the stereoselective reduction of ω-ketoesters.8 In
seeking to expand the substrate repertoire for CaADH, we set
out to challenge this enzyme with a battery of β-
ketophosphonates as a new and highly value-added substrate
class. Indeed, we are delighted to report here that CaADH
enantioselectively reduces an expansive array of α,α-difluori-
a
Yields (in black) represent isolated quantities of pure secondary
alcohols; enantiomeric excesses (in teal) determined by chiral phase-
HPLC; D-glucose employed as terminal reductant throughout, utilizing
NADP+-dependent D-glucose DH from Thermoplasma acidophilum.
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To our knowledge, these represent the first examples of a
dehydrogenase-based entry into valuable enantioenriched α,α-
difluorinated-β-hydroxyphosphonates. Stereoselective ap-
proaches to β-hydroxyphosphonates, particularly those bearing
α-fluorination, have been quite limited so far. A handful of
enzymatic approaches based upon kinetic resolution exist, but
have been limited to simple systems with limited function-
ality.38 Chemical routes include the asymmetric reduction of β-
keto phosphonates with pinene-derived chiral organoboron
reagents, a borohydride/chiral auxiliary combination, or via
Ru(II)-BINAP-mediated hydrogenation.39 Of these, only the
latter asymmetric hydrogenation approach of Kitamura and
Noyori provides significant stereoinduction.39
for the reduction of β-keto-carboxylate esters8 (Figures 2 and
3). To investigate this further, computational docking experi-
CaADH Active Site Plasticity: High Enantioselectivity,
Yet Opposite Facial Selectivity for α-Difluorinated β-
Keto-phosphonate Substrates vs ω-Keto-carboxylate
Substrates. In order to determine the absolute stereo-
chemistry of the CaADH-derived alcohols obtained here, the
Mosher ester method was initially applied, utilizing alcohols 2d
and 2f. According to the Kakisawa model, as is shown in Figure
2 for the case of 2f, the alcohol stereochemistry is predicted to
Figure 3. CaADH: Substrate promiscuity with stereochemical fidelity.
(Top) β-Keto carboxylate ester reduced with D-stereochemistry as
evidenced by HPLC on Chiralcel OD vs racemic standard (90:10
hexane/i-PrOH; 1 mL/min). (Bottom) β-Keto-α,α-difluorophospho-
nate ester reduced with L-stereochemistry; also Chiralcel OD-HPLC
(95:5 hexane/i-PrOH).
ments were performed in the CaADH active site with a
representative β-keto ester substrate, ethyl benzoylacetate, and
with a typical α,α-difluoro-β-ketophosphonate 1f (Figure 4;
Alignment: Clustal W;41 Homology Modeling: MODELLER;42
Molecular Dynamics: GROMACS;43 Docking: Autodock 4;44
Graphics/Rendering: VMD45; see Supporting Information for
details). The result suggests that both substrates occupy the
same binding pocket of the enzyme, even though the facial
preference for hydride transfer is reversed. Ethyl benzoylacetate
docks with its keto group coordinated to S140, a typical binding
motif for short-chain dehydrogenases.46 This orientation gives
rise to the D-stereoisomer. However, the keto-phosphonate 1f
docks with its keto group inverted, driven by a favorable H-
bonding interaction with K207. This difference in keto
orientation would give rise to the L-stereoisomer, as observed.
This type of substrate inversion contrasts with typical cases of
reversed enantioselectivity in enzyme active sites, which
normally arise from alteration of substituent size that, in turn,
transposes the occupants of medium and large active site
pockets.47
Figure 2. Determination of the absolute stereochemistry of the
CaADH-derived products. (Top) Mosher ester analysis for 2f.
(Bottom) X-ray crystallographic structures for 2f and 2h.
be L.40 Pleasingly, X-ray crystallographic structural determi-
nation for alcohols 2f and 2h utilizing anomalous dispersion
served to confirm the L-stereochemistry (Figure 2, bottom).
Molecular Modeling: Exploring the Origins of CaADH
Active Site Plasticity. CaADH has proven to be an
exceptionally versatile enzyme from the point of view of
asymmetric synthesis, being able to process substrates that
range from (i) a broad variety of ω-keto carboxylate esters8 to
(ii) the significant spectrum of α,α-difluorinated β-keto
phosphonate esters examined here (Scheme 2). That said, the
facial selectivity observed for the CaADH reduction of β-keto-
phosphonate esters here is opposite to that previously observed
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precise molecular details of enzyme−substrate recognition here,
going forward, there will be a serious effort to crystallize
CaADH, both as the holoenzyme and in ketone-bound states.
Ultimately, such structural biological studies will be invaluable
in further addressing the question of how CaADH catalyzes the
reduction of these two substrate classes. It should be noted that
there are really three structural changes seen in moving from
the D- to the L-selective substrates depicted in Figure 4. (i)
While perhaps the most notable change is the replacement of
the carboxylate ester with a phosphonate ester, (ii) α-
fluorination is also introduced and (iii) a double bond is also
inserted (phenyl to styrenyl). Future studies will be directed at
mapping how the observed switch in enantioselectivity evolves
across this set of structural changes. All told, this is a notable
example of substrate promiscuity with stereochemical fidelity, but
with an interesting twist; namely, antipodal facial preference
based upon compound class (Figure 4).47 CaADH displays re-face
hydride delivery (D-product) to β-keto carboxylate esters, but
pseudo-si-face hydride delivery (L-product) to β-keto fluori-
nated phosphonate esters. That the CaADH apparently exhibits
complementary modes of binding two different substrate
classes, and yet processes each with high efficiency and
stereochemical fidelity, speaks to the unusual active site
plasticity of this enzyme.
Thiono-Claisen Rearrangements: Structure−Reactiv-
ity Studies. With the difluorinated-β-hydroxyphosphonates, in
hand, we next set out to investigate the desired [3,3]-
sigmatropic rearrangement. Toward this end, the cinnamyl
substrate, 2f, was chosen, and four distinct thiono-esters were
synthesized; namely (i) the xanthate; (ii) the phenyl carbonate;
(iii) the pentafluorophenyl thionocarbonate, and (iv) the N-
phenyl thionocarbamate (Figure 4). To study the rearrange-
ment of each, advantage was taken of the difluorinated
phosphonate moiety permitting the kinetics of rearrangement
to be conveniently followed by 19F NMR (Scheme 3). It
quickly became clear that the xanthate, though able to undergo
the targeted rearrangement at room temperature, did so at a
very slow rate, with the observed half-time being approximately
2.5 days. On the other hand, replacing the SMe group with the
more electronegative OPh, NHPh, and OC6F5 groups led to a
rather dramatic structure−reactivity relationship (SRR) with
the half-time decreasing significantly across this series.
Most notable is the pronounced effect of distal fluorination
upon rate, whereby the simple expedient of moving from an
allylic phenyl thionocarbonate to the corresponding pentafluoro-
phenyl thionocarbonate accelerates the sigmatropic rearrange-
ment by a factor of 400 [half-time goes from 20 h to 3 min
(0.05 h)]. Note that we use the term “distal” in consideration
that all substituent effects seen here are for substitution upon
an aromatic nucleus that is not in direct resonance with the six-
membered transition state, as would be the case for a
thionobenzoate system, for example. Rather, here an oxygen
atom serves as a bridge between the arene ring and the atoms of
the six-centered system for the sigmatropic rearrangement.
Moreover, given the electronically opposing inductive and
resonance effects of fluorine substitution, the significant rate
enhancement seen here by fluorination of this distal aromatic
nucleus is really quite notable and could not have been
anticipated prior to conducting these studies.
Figure 4. Active site plasticity: a view from molecular modeling. The
observed switch in facial selectivity as a function of substrate class with
preservation of high facial selectivity is evidence of unusual active site
plasticity in CaADH. Note: The coordinates of the NADPH-cofactor
(background) were imported from PDB-1XG5.
The model suggested by the experimental results and the
supporting docking studies here will drive future experimenta-
tion. Note that the docked, chain-extended conformer shown in
Figure 4 was selected by visual inspection as the most
reasonable member of a set of 19 conformers in the lowest
energy cluster identified by molecular docking with Autodock
4.44 Factors that were considered in inspecting cluster members
were NADPH-C4′-substrate carbonyl distance and positioning
with respect to hydride transfer, as well as consistency with the
known stereochemical outcome of hydride transfer. There is
some variation among docked conformers in the rotamer
distribution about the Cα−Cβ bond, with some deviation from
the antiarrangement of the phosphonate and Cγ when sighting
down this bond. This is perhaps to be expected given the
known tendency of α-fluorine substitution to elicit Thorpe−
Ingold-like effects in some systems.48 To shed light on the
Indeed, a survey of the literature reveals that the rate
observed for this allylic pentafluorophenyl thionocarbonate
rearrangement (t1/2 ≈ 3 min @ rt) is among the fastest seen for
a neutral (hetero)Claisen rearrangement (see the Supporting
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at elevated temperature is converted to a room temperature
reaction by judicious placement of an oxyanionic substituent in
the earlier cases, and by judicious introduction of fluorination
into an O-aryl substituent here.
Scheme 3. Structure−Reactivity Relationships across a
a
Family of Thiono-Claisen Rearrangements
Thiono-Claisen Rearrangement: Scope and Stereo-
chemical Course. To examine the scope and stereochemical
fidelity, the rearrangement of pentafluorophenyl allylic
thionocarbonates was next examined across the battery of
structurally diverse, enantiomerically enriched substrates
generated via CaADH-mediated β-keto-phosphonate reduction.
As is depicted in Scheme 4, the desired [3,3]-sigmatropic
Scheme 4. Substrate Scope for the New Allylic
Pentafluorophenyl Thionocarbonate Rearrangement
a
a
(a) Synthetic schemes and measured half-times for the four thiono-
Claisen rearrangements studied here. (b) 19F NMR stack plot for the
rearrangement of the pentafluorophenyl thionocarbonate ester @ rt.
Note: The t = 0 point is arbitrarily labeled; this rearrangement
proceeds too fast to capture a true starting time point.
Information for comprehensive tables). Earlier studies49 noted
that properly situated alkoxy groups could accelerate the parent
Claisen rearrangement by up to 2 orders of magnitude. The
most facile rearrangement seen in those studies has a half-time
of 45 min @ room temperature (alkoxy substitution;
cyclopentenyl ether). Similarly, the Ireland−Claisen rearrange-
ment (silyloxy substituent) proceeds with t1/2 = 1−210 min @
42 °C.50 Probably the most well studied Claisen variant with an
additional heteroatom, the allylic imidate rearrangement,
proceeds with t1/2 = 45 min at 140 °C.51
Of course, charge-accelerated [3,3]-sigmatropic rearrange-
ments have drawn great attention in the synthetic and
mechanistic chemistry communities, as these are widely
regarded as among the most facile such rearrangements
known, with enormous rate enhancements reported for oxy-
anionic variants of the Cope and Claisen rearrangement, for
example.10,52 Probably the key finding here is that, in terms of
absolute rate, the title allylic pentafluorophenyl thionocarbon-
ate rearrangement (t1/2 = 3 min @ 23 °C) is on par with (i) the
anionic oxy-Cope rearrangement reported by Evans and Golob
(t1/2 = 8 min @ 0 °C; K+ counterion, 18-Cr-6)10 and (ii) the
anionic oxy-Claisen rearrangement discovered by Koreeda a
decade later (t1/2 = 6 min @ −23 °C; K+ counterion).52 In all
three cases, a sigmatropic rearrangement that normally occurs
a
Yields (in black) represent isolated quantities of pure secondary
alcohols; enantiomeric excesses (in green) determined by chiral phase-
HPLC. *This RR requires mild heating (50 °C); all others proceed
smoothly at rt.
rearrangement proceeds seamlessly across the series, with
essentially complete conservation of ee in transposing the β-
hydroxy stereocenters in the educts into a δ-thio stereocenter in
the products. The enantiomeric excess was determined by
chiral phase HPLC analysis, and the absolute stereochemistry
of the rearrangement product was unambiguously established in
the case of product 3f via chemical correlation. Namely,
ozonolytic processing of 3f was followed by comparison of the
measured optical rotation obtained for the methyl (S)-
thiomandelate oxidation/esterification product thereby ob-
tained to the literature value (see Supporting Information for
details). Diverse functionality is tolerated, both in the active site
and in the subsequent rearrangement from aromatic hetero-
cycles and fluorinated phosphonates (all cases) to benzyl ether
and orthobicyclo[2.2.2]octyl (OBO) ester protecting groups.
Indeed, especially noteworthy is the ability to carry a bulky
OBO ester through both the enzymatic reduction and
sigmatropic rearrangement steps. The OBO ester neither
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perturbs the exquisite facial selectivity observed in the CaADH
active site nor blocks the rearrangement, although the sizable
orthoester is adjacent to the C−S bond-forming center. As
noted at the outset, the resultant product, 3e, presents an ideal
platform upon which to build a focused library of Zn-
aminopeptidase inhibitor candidates.21 The essentially com-
plete conservation of ee likely results from a pericyclic process
that proceeds through a chairlike transition state, with both the
α,α-difluorinated phosphonate ester and the R-group at the δ-
position disposed pseudoequatorially. However, given the
precedent for boat-like transition states in certain Claisen-
type rearrangements,53 most notably of the Ireland variety, at
this stage one cannot rule out a boatlike transition state in
which the δ-R group is disposed in the bowsprit position, for
example.
Eyring Analysis and Solvent Dependence. While the
low activation barrier, functional group tolerance, and exquisite
stereocontrol observed constitute the practical advantages of
the title transformation, perhaps even more important from a
reaction design standpoint is the observation that one gains 2 to
3 orders of magnitude in rate enhancement via distal fluorine
substitution here, as discussed. This remarkable SRR prompted
us to investigate how fluorination leads to a more favorable free
energy of activation (ΔΔG‡ ≈ 3.6 kcal/mol) for this type of
sigmatropic rearrangement. Accordingly, Eyring analysis was
performed on three different classes of thiono-ester to obtain
the enthalpic and entropic contributions for each rearrange-
ment (Figure 5). The results yield an observed enthalpy of
rearrangements in general, ΔS‡ is almost always negative,
presumably reflecting the need for a precisely organized
transition state. Indeed, studies of the Claisen and related
rearrangements reveal just this: (i) cyclopentenyl allyl ether
(−21 e.u);49 (ii) (uncatalyzed) chorismate rearrangement (−13
e.u.);56 (iv) allylic imidate rearrangement (−18 e.u.); (v) allylic
thionobenzoate rearrangement (−10 e.u.).55 Prior to these
findings, we are aware of only a couple of cases in which a
favorable entropy of activation has been observed for a neutral
[3,3]-sigmatropic rearrangement in the absence of a catalyst,
the most notable involving a system in which the terminal
alkene is masked as a cyclopropane,54 i.e. technically not a
bonafide example of a [3,3]-sigmatropic rearrangement.
To further understand the transition state, the rearrangement
of 2f was carried across a number of solvents. A rate
enhancement is observed as the solvent dielectric constant
increases as shown in Figure 6. This solvent dependence upon
Figure 6. Rate dependence upon solvent-rearrangement kinetics of 2f.
The increase in kobs as a function of solvent dielectric is suggestive of a
polarized, partially dissociative transition state (lower right) as
opposed to a symmetrical, synchronous one (upper right).
rate trends similarly to, but appears to be somewhat more
pronounced than, that observed in the aforementioned
thionobenzoate system.57 Interestingly then, by moving from
THF to acetonitrile, one accelerates the title rearrangement still
further, with t1/2 ≈ 1.7 min @ rt in the latter solvent. This
solvent dependence, when combined with the stereochemical
course discussed above, suggests that the transition state for the
title rearrangement is significantly more polarized than the
ground state, i.e. partially dissociative as depicted in Figure 6,
yet still chairlike, as depicted in Scheme 5.
CONCLUSIONS
■
As was mentioned at the outset, we have entered an age in
which the state-of-the-art solution for complex synthesis,
chemical biology, or process chemistry may involve the careful
melding of sophisticated enzymatic chemistry with sophisti-
cated organic chemistry.58 This study describes the melding of a
dehydrogenase enzyme from Clostridium acetobutylicum
(CaADH) that exhibits remarkable active site plasticity with a
new allylic pentafluorophenyl thionocarbonate rearrangement
that is among the fastest sigmatropic rearrangements yet
reported (t1/2 = 1.7 min at rt in MeCN). The combination of
(i) LiCF2P(O)(OR)2/ester condensation, (ii) highly stereo-
selective CaADH-mediated α,α-difluoro-β-keto-phosphonate
Figure 5. Eyring analysis yields compartitive enthalpies and entropies
of activation for several thiono-Claisen RR types.
activation (ΔH‡) for rearrangement ranging from 24 to 27
kcal/mol for all three thiono-Claisen rearrangements. This is
consistent with values observed for other [3,3]-sigmatropic
rearrangements.51,54,55
However, the entropy of activation (ΔS‡) was found to be
highly favorable for both thionocarbonates examined and for
the xanthate. This is quite unusual. As noted, for sigmatropic
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active site. Based upon molecular docking, a model is put forth
here to account for these complementary binding modes (i.e.,
switch of key catalytic residues for carbonyl protonation from
S140 to K207) and will drive structural biology studies to better
understand this very promising biocatalyst.
Scheme 5. Transition State Geometry for the Stereospecific
[3,3]-Sigmatropic Rearrangement
a
For the new sigmatropic rearrangement, Eyring analysis
indicates a favorable entropy of activation for the title
transformation (14 e.u.) that contrasts with values reported
for most [3,3]-sigmatropic rearrangements. The highly stereo-
retentive nature of the rearrangement points to an organized,
pericyclic transition state (Scheme 3), and the preference for
polar solvents suggests that this transition state is also polarized
and asynchronous (Figure 6). That said, the large positive
entropy of activation observed cannot be accounted for solely
by a polarized transition state that must remain organized to
seamlessly transmit stereochemistry from educt to product.
One could imagine that another source of entropy gain must be
at play here, such as release of a solvent molecule or relaxation
of solvent structure along the reaction coordinate as one
approaches the transition state. Clearly, the findings reported
here will drive future computational studies to better
understand the geometry and charge distribution in the
transition state for the title rearrangement.
Perhaps the most notable observation here is that this
exceptionally facile sigmatropic rearrangement displays a distal
fluorine effect that includes both a significant enthalpic
component (ΔΔH‡ = −2.4 kcal/mol) and a significant entropic
component (ΔΔS‡ = 4 e.u.; 1.2 kcal/mol @ rt). These
enthalpic and entropic effects arise exclusively from fluorination
of the distal heteroatom-bridged arene in the thionocarbonate
substrate. While one can imagine that such fluorination might
help to dissipate charge in the likely polarized transition state,
one cannot easily intuit how this would parse into enthalpic and
entropic components; computational interrogation will be
needed. That said, these observations speak to the power of
fluorine substitution to drive this particular thiono-Claisen
rearrangement and raise the possibility that such a strategy may
be able to drive a broader suite of pericyclic reactions in the
future.
a
Consideration of possible transition state conformations for the facile,
stereospecific sigmatropic RR of allylic pentafluorophenyl thionocar-
bonates described herein. Note: All conformations shown are
consistent with the observed stereochemistry at the newly formed
C−S bond, but only the O,CsB and CsCO transition states also give the
correct alkene geometry.
reduction, and (iii) pentafluorophenyl thionocarbonate sigma-
tropic rearrangement provides a highly efficient, stereo-
controlled entry into densely functionalized fluorinated
phosphonate synthons of value to chemical biology. In
particular, a platform for the construction of a focused library
of zinc aminopeptidase A-directed inhibitor candidates is
constructed here. Importantly, this example also illustrates the
value in challenging enzymes with complex, advanced
substrates. In this case, the enzymatic substrate features 12
carbon atoms and an additional 10 heteroatoms and includes an
unusual bridged oxabicyclo[2.2.2]octyl ring system; yet, it is
processed efficiently by the CaADH enzyme (79% isolated
yield, 99% ee).
As for the enzymology, in the Introduction, it was pointed
out that, from the point of view of asymmetric synthesis, one
really seeks biocatalysts that show both high enantioselectivity
and broad substrate generality,59,60 whether these enzymes be
“engineered” through Darwinian evolution in the biosphere,4
through “directed evolution” in the laboratory,5,59,61 or through
de novo design in silico.6,62 Only then can one associate that
enzyme with a truly reliable “biocatalytic retrosynthetic1
transform arrow.”3 To put the observed “active site plasticity”
into perspective, it is well to compare the CaADH enzyme to
literature precedents for enzymes of value to synthesis. For
example, Kroutil and co-workers describe a dehydrogenase
from Ralstonia that also shows the ability to process distinct
carbonyl compound classes and does so with a switch in
stereofacial preference, suggestive of distinct binding modes,
one for aryl alkyl ketones, another for α- or β-keto esters.63 Yet,
with this plasticity, there is a drop-off in enantioselectivity in
going from enzymatic reduction of the former substrate class
(99% ee, six examples) to the latter (96%, 64%, 37% ee, three
examples). Thus, the fact that high stereoselectivity is seen in
both the binding mode for ω-keto carboxylate esters (90−99%
ee), producing L-products,8 and also for β-keto-α,α-difluor-
ophosphonate esters (93−99% ee), producing D-products,
attests to a particularly fortuitous plasticity in this CaADH
EXPERIMENTAL SECTION
■
Ester Addition Route into β-Keto-α,α-difluorophosphonates
(Aryl Systems). Aryl-linked α,α-difluoro-β-keto phosphonates were
efficiently synthesized by the addition of lithiated α,α-difluoro-
phosphonate to the α,β-unsaturated methyl esters. The methyl ester
substrates were synthesized via Horner−Wadsworth−Emmons
condensation chemistry. A typical procedure for the PCF2−C-bond
forming reaction, using 1j as an example, is as follows. To
diisopropylamine (297 μL, 2.12 mmol, 2 equiv) in THF at −78 °C
under a N2 atmosphere was added n-BuLi (1.33 mL, 2.12 mmol, 2
equiv, 1.6 M in hexanes), dropwise via syringe. The resulting solution
was allowed to warm to 0 °C for 25 min and then cooled to −78 °C,
followed by dropwise addition of diethyl α,α-difluoromethylphos-
phonate (298 mg, 1.59 mmol, 1.5 equiv) dissolved in THF. After 30
min at −78 °C, the benzofuran-substituted α,β-unsaturated methyl
ester (215 mg, 1.06 mmol, 1 equiv) in THF was added slowly to the
reaction mixture via syringe down the wall of the flask. The reaction
mixture was left for 1−2 h at the same temperature and then quenched
with a few drops of conc. acetic acid. The reaction mixture was
gradually brought to room temperature, diluted with NH4Cl (aq), and
extracted with EtOAc. The combined organics were dried over sodium
sulfate, filtered, and concentrated. Purification by column chromatog-
raphy (typically eluent gradient 10−30% EtOAc in hexanes) yielded
the product 1j (365 mg, 96%).
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Aldehyde Addition/DMP Oxidation Route into β-Keto-α,α-
difluoro-phosphonates (Alkyl Systems). The general procedure
for the two-step synthesis of the β-keto-α,α-difluoro-phosphonate
substrates proceeded as follows (using 1c as an example): To
diisopropylamine (2.80 mL, 20.0 mmol, 2 equiv) in THF at −78 °C
under a nitrogen atmosphere was added n-BuLi (12.5 mL, 20.0 mmol,
2 equiv, 1.6 M in hexanes), dropwise via syringe. The resulting
solution was allowed to warm to 0 °C for 25 min and then cooled to
−78 °C, followed by dropwise addition of diethyl α,α-difluoromethyl-
phosphonate (3.76 g, 20.0 mmol, 2 equiv) dissolved in THF. After 30
min at −78 °C, crotonaldehyde (700 mg, 9.98 mmol, 1 equiv) in THF
was added slowly to the reaction mixture via syringe down the wall of
the flask. The reaction mixture was left for 2 h at the same temperature
and then quenched with aqueous NH4Cl solution and gradually
brought to room temperature. The reaction mixture was extracted with
EtOAc. The combined organics were dried over sodium sulfate,
filtered, and concentrated. If necessary, the α,α-difluoro-β-hydroxy-
phosphonate intermediate was purified by column chromatography.
The β-hydroxyphosphonate obtained (2.59 g, 9.98 mmol, 1 equiv) was
dissolved in dry dichloromethane under a nitrogen atmosphere and
was cooled to 0 °C, followed by the addition of Dess-Martin
periodinane (5.50 g, 12.97 mmol, 1.3 equiv) in one portion. The
reaction mixture was stirred at the same temperature for 15 min,
brought to room temperature, and stirred for an additional 2 h.
Progress of the reaction was monitored by TLC. The reaction mixture
was diluted with saturated sodium bicarbonate solution and extracted
with dichloromethane. The organic layer was dried with sodium
sulfate, filtered, and concentrated. In this case, purification by column
chromatography (typically eluent gradient 10−30% EtOAc in hexanes)
yielded β-keto-α,α-difluorophosphonate 1c in (2.25 g, 88%).
Enzyme Expression, Purification, and Standardization.
CaADH was expressed and purified as previously described. CaADH
was standardized by monitoring the reduction of benzaldehyde by
NADPH at 340 nm. One unit is defined as the amount of enzyme
needed to reduce 1 μmol of benzaldehyde to benzyl alcohol in 1 min.
Enzymatic β-Keto-α,α-Difluoro-phosphonate Reductions.
The general procedure for the enzymatic reductions (using 2f as an
example) is as follows. A solution of NADP+ (17 mg, 0.02 mmol, 0.01
equiv), D-glucose (4.0 g, 22.3 mmol, 10 equiv), CaADH (10 units),
and glucose dehydrogenase (2 units)(Thermoplasma acidophilum) in
KPO4 buffer (100 mM, pH 7.0) was shaken at 37 °C and 250 rpm.
Keto-phosphonate 1f (730 mg, 2.29 mmol, in DMSO) was added so
that the final DMSO concentration was 10% (v/v). Reaction progress
was monitored by TLC until conversion was complete. Product was
extracted with ethyl acetate and dried over sodium sulfate. Following
vacuum filtration and concentration, purification by column
chromatography (typically eluent gradiet 20−40% EtOAc in hexanes)
yielded product 2f (675 mg, 92%). Alcohol products were further
characterized by NMR and chiral HPLC (see Supporting Information
for details).
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures; characterization of new
compounds, including copies of NMR spectra; kinetic data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*dberkowitz1@unl.edu
Author Contributions
§K.P. and G.A.A. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Douglas R. Powell (U. of Oklahoma) for X-
ray crystal structure determination and the NSF (1214019) for
support. This research was facilitated by the IR/D (Individual
Research and Development) program associated with D.B.B.’s
appointment at the NSF. The authors thank the NIH (SIG-1-
510-RR-06307) and NSF (CHE-0091975, MRI-0079750) for
NMR instrumentation support and the NIH (RR016544) for
facilities renovation.
REFERENCES
■
(1) Turner, N. J.; O’Reilly, E. Nat. Chem. Biol. 2013, 9, 285−288.
(2) Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.;
Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J. Science
2010, 329, 305−309.
(3) (a) Corey, E. J.; Long, A. K.; Lotto, G. I.; Rubenstein, S. D. Recl.
Trav. Chim. Pays-Bas 1992, 111, 304−10. (b) Corey, E. J.; Jorgensen,
W. L. J. Am. Chem. Soc. 1976, 98, 203−9.
(4) Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232−240.
(5) Bornscheuer, U.; Huisman, G.; Kazlauskas, R.; Lutz, S.; Moore, J.;
Robins, K. Nature 2012, 485, 185−194.
(6) Siegel, J. B.; Zanghellini, A.; Lovick, H. M.; Kiss, G.; Lambert, A.
R.; St. Clair, J. L.; Gallaher, J. L.; Hilvert, D.; Gelb, M. H.; Stoddard, B.
L.; Houk, K. N.; Michael, F. E.; Baker, D. Science 2010, 329, 309−313.
(7) Yoon, T. P.; Jacobsen, E. N. Science 2003, 299, 1691−1693.
(8) Applegate, G. A.; Cheloha, R. W.; Nelson, D. L.; Berkowitz, D. B.
Chem. Commun. 2011, 47, 2420−2422.
(9) (a) Obexer, R.; Studer, S.; Giger, L.; Pinkas, D. M.; Gruetter, M.
G.; Baker, D.; Hilvert, D. ChemCatChem 2014, 6, 1043−1050.
(b) Bacik, J.-P.; Whitworth, G. E.; Stubbs, K. A.; Vocadlo, D. J.; Mark,
B. L. Chem. Biol. 2012, 19, 1471−1482. (c) Muralidhara, B. K.; Sun, L.;
Negi, S.; Halpert, J. R. J. Mol. Biol. 2008, 377, 232−245. (d) Todd, A.
E.; Orengo, C. A.; Thornton, J. M. Trends Biochem. Sci. 2002, 27, 419−
426.
(10) Evans, D. A.; Golob, A. M. J. Am. Chem. Soc. 1975, 97, 4765−6.
(11) (a) Balavoine, F.; Azizi, M.; Bergerot, D.; Mota, N.; Patouret, R.;
Roques, B. P.; Llorens-Cortes, C. Clin. Pharmacokinet. 2014, 53, 385−
395. (b) Marc, Y.; Gao, J.; Balavoine, F.; Michaud, A.; Roques, B. P.;
Llorens-Cortes, C. Hypertension 2012, 60, 411−418. (c) Padia, S. H.;
Kemp, B. A.; Howell, N. L.; Fournie-Zaluski, M.-C.; Roques, B. P.;
Carey, R. M. Hypertension 2008, 51, 460−465. (d) Fournie-Zaluski,
M.-C.; Fassot, C.; Valentin, B.; Djordjijevic, D.; Reaux-Le Goazigo, A.;
Corvol, P.; Roques, B. P.; Llorens-Cortes, C. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 7775−7780.
Thiono-Claisen Sigmatropic Rearrangement. A typical thiono-
Claisen RR is carried out as follows, using 2j as an example. To alcohol
2j (191 mg, 0.53 mmol, 1 equiv) in THF at −78 °C was added n-BuLi
(364 μL, 0.58 mmol, 1.1 equiv, 1.6 M in hexanes) under an inert
atmosphere (N2). After 15 min, pentafluorophenyl thionochloro-
formate (119 μL, 0.742 mmol, 1.4 equiv) was added dropwise, via
syringe, and the reaction was allowed to run for 30−60 min. The
reaction was quenched with an aqueous, saturated NH4Cl solution.
The mixture was extracted with Et2O. The combined organics were
dried (Na2SO4), filtered, concentrated, and left at room temperature, if
necessary, until the rearrangement was complete. The rearrangement
is easily followed by TLC, as one generally observes conversion of the
thionocarbonate intermediate to a more polar thiocarbonate product.
The product thiocarbonate was purified by column chromatography
(typically eluent gradient 10−30% EtOAc in hexanes) to yield
thiocarbonate 3j (292 mg, 94%).
(12) Fei, X.; Holmes, T.; Diddle, J.; Hintz, L.; Delaney, D.; Stock, A.;
Renner, D.; McDevitt, M.; Berkowitz, D. B.; Soukup, J. K. ACS Chem.
Biol. 2014, 9, 2875−2882.
(13) (a) Loranger, M. W.; Forget, S. M.; McCormick, N. E.; Syvitski,
R. T.; Jakeman, D. L. J. Org. Chem. 2013, 78, 9822−9833. (b) Diab, S.
3608
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Journal of the American Chemical Society
Article
A.; De Schutter, C.; Muzard, M.; Plantier-Royon, R.; Pfund, E.;
Lequeux, T. J. Med. Chem. 2012, 55, 2758−2768.
(14) (a) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.;
Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. J.
Am. Chem. Soc. 2010, 132, 7617−7625. (b) Nieschalk, J.; O’Hagan, D.
J. Chem. Soc., Chem. Commun. 1995, 719−720.
(15) Berkowitz, D. B.; Bose, M.; Pfannenstiel, T. J.; Doukov, T. J.
Org. Chem. 2000, 65, 4498−4508.
(16) Berkowitz, D. B.; Eggen, M.; Shen, Q.; Shoemaker, R. K. J. Org.
Chem. 1996, 61, 4666−4675.
(40) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092−6.
(41) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Nucleic Acids Res.
1994, 22, 4673−80.
(42) Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779−815.
(43) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem.
Theory Comput. 2008, 4, 435−447.
(44) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,
R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785−
2791.
(45) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996,
(17) (a) Romanenko, V. D.; Kukhar, V. P. Chem. Rev. 2006, 106,
3868−3935. (b) Lopin, C.; Gautier, A.; Gouhier, G.; Piettre, S. R. J.
Am. Chem. Soc. 2002, 124, 14668−14675. (c) Lequeux, T. P.; Percy, J.
M. J. Chem. Soc., Chem. Commun. 1995, 2111−2112.
(18) Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc.,
Chem. Commun. 1981, 930−932.
14, 33−38.
(46) Kallberg, Y.; Oppermann, U.; Jornvall, H.; Persson, B. Eur. J.
Biochem. 2002, 269, 4409−4417.
(47) Mugford, P. F.; Wagner, U. G.; Jiang, Y.; Faber, K.; Kazlauskas,
R. J. Angew. Chem., Int. Ed. 2008, 47, 8782−8793.
́
(48) Urbina-Blanco, C. A.; Skibinski, M.; O’Hagan, D.; Nolan, S. P.
(19) McKenna, C. E.; Shen, P.-D. J. Org. Chem. 1981, 46, 4573−
4576.
Chem. Commun. 2013, 49, 7201−7203.
(49) Coates, R. M.; Rogers, B. D.; Hobbs, S. J.; Curran, D. P.; Peck,
D. R. J. Am. Chem. Soc. 1987, 109, 1160−70.
(20) (a) Panigrahi, K.; Eggen, M.; Maeng, J.-H.; Shen, Q.; Berkowitz,
D. B. Chem. Biol. 2009, 16, 928−936. (b) Szewczuk, L. M.; Tarrant, M.
K.; Sample, V.; Drury, W. J.; Zhang, J.; Cole, P. A. Biochemistry 2008,
47, 10407−10419.
(21) David, C.; Bischoff, L.; Meudal, H.; Mothe, A.; De Mota, N.;
DaNascimento, S.; Llorens-Cortes, C.; Fournie-Zaluski, M.-C.;
Roques, B. P. J. Med. Chem. 1999, 42, 5197−5211.
(50) Ireland, R. E.; Mueller, R. H.; Willard, A. K. J. Am. Chem. Soc.
1976, 98, 2868−77.
(51) Overman, L. E. J. Am. Chem. Soc. 1976, 98, 2901−10.
(52) Koreeda, M.; Luengo, J. I. J. Am. Chem. Soc. 1985, 107, 5572−3.
(53) (a) Gul, S.; Schoenebeck, F.; Aviyente, V.; Houk, K. N. J. Org.
Chem. 2010, 75, 2115−2118. (b) Chen, C.-L.; Namba, K.; Kishi, Y.
Org. Lett. 2009, 11, 409−412.
(22) Christensen, B. G.; Schmitt, S. M.; Johnston, D. B. R.;
Application: EP-1985-108135; 167139, 1986; p 185.
(23) Singer, A. G.; Ghomashchi, F.; Le Calvez, C.; Bollinger, J.;
Bezzine, S.; Rouault, M.; Sadilek, M.; Nguyen, E.; Lazdunski, M.;
Lambeau, G.; Gelb, M. H. J. Biol. Chem. 2002, 277, 48535−48549.
(24) (a) Sengupta, R. N.; Herschlag, D.; Piccirilli, J. A. ACS Chem.
Biol. 2012, 7, 294−299. (b) Sun, S.; Yoshida, A.; Piccirilli, J. A. RNA
1997, 3, 1352−1363. (c) Piccirilli, J. A.; Vyle, J. S.; Caruthers, M. H.;
Cech, T. R. Nature 1993, 361, 85−8.
(54) Harano, K.; Kiyonaga, H.; Hisano, T. Chem. Pharm. Bull. 1987,
35, 1388−96.
(55) McMichael, K. D. J. Am. Chem. Soc. 1967, 89, 2943−7.
(56) Jackson, D. Y.; Liang, M. N.; Bartlett, P. A.; Schultz, P. G.
Angew. Chem., Int. Ed. 1992, 31, 182−3.
(57) Smith, S. G. J. Am. Chem. Soc. 1961, 83, 4285−4287.
(58) (a) Kohler, V.; Turner, N. J. Chem. Commun. 2015, 51, 450−
464. (b) Heidlindemann, M.; Rulli, G.; Berkessel, A.; Hummel, W.;
Groeger, H. ACS Catal. 2014, 4, 1099−1103. (c) Reetz, M. T. J. Am.
Chem. Soc. 2013, 135, 12480−12496. (d) Dunsmore, C. J.; Carr, R.;
Fleming, T.; Turner, N. J. J. Am. Chem. Soc. 2006, 128, 2224−2225.
(59) Bornscheuer, U. T.; Kazlauskas, R. J. Angew. Chem., Int. Ed.
2004, 43, 6032−6040.
(60) Carr, R.; Alexeeva, M.; Enright, A.; Eve, T. S. C.; Dawson, M. J.;
Turner, N. J. Angew. Chem., Int. Ed. 2003, 42, 4807−4810.
(61) (a) Wu, Q.; Soni, P.; Reetz, M. T. J. Am. Chem. Soc. 2013, 135,
1872−1881. (b) Daugherty, A. B.; Govindarajan, S.; Lutz, S. J. Am.
Chem. Soc. 2013, 135, 14425−14432. (c) Abrahamson, M. J.; Vazquez-
Figueroa, E.; Woodall, N. B.; Moore, J. C.; Bommarius, A. S. Angew.
Chem., Int. Ed. 2012, 51, 3969−3972. (d) Reetz, M. T. Angew. Chem.,
Int. Ed. 2011, 50, 138−174. (e) Musa, M. M.; Phillips, R. S. Catal. Sci.
Technol. 2011, 1, 1311−1323. (f) Reetz, M. T.; Prasad, S.; Carballeira,
J. D.; Gumulya, Y.; Bocola, M. J. Am. Chem. Soc. 2010, 132, 9144−
9152. (g) Toscano, M. D.; Woycechowsky, K. J.; Hilvert, D. Angew.
Chem., Int. Ed. 2007, 46, 3212−3236. (h) Kazlauskas, R. J. Curr. Opin.
Chem. Biol. 2005, 9, 195−201. (i) Qian, Z.; Lutz, S. J. Am. Chem. Soc.
2005, 127, 13466−13467. (j) May, O.; Nguyen, P. T.; Arnold, F. H.
Nat. Biotechnol. 2000, 18, 317−320.
(25) Meena; Sam, M.; Pierce, K.; Szostak, J. W.; McLaughlin, L. W.
Org. Lett. 2007, 9, 1161−1163.
(26) Claisen, L. Ber. Dtsch. Chem. Ges. 1913, 45, 3157−66.
(27) Luk, L. Y. P.; Qian, Q.; Tanner, M. E. J. Am. Chem. Soc. 2011,
133, 12342−12345.
(28) McIntosh, J. A.; Donia, M. S.; Nair, S. K.; Schmidt, E. W. J. Am.
Chem. Soc. 2011, 133, 13698−13705.
(29) Hilvert, D.; Nared, K. D. J. Am. Chem. Soc. 1988, 110, 5593−4.
(30) Jackson, D. Y.; Jacobs, J. W.; Sugasawara, R.; Reich, S. H.;
Bartlett, P. A.; Schultz, P. G. J. Am. Chem. Soc. 1988, 110, 4841−2.
(31) Pervushin, K.; Vamvaca, K.; Voegeli, B.; Hilvert, D. Nat. Struct.
Mol. Biol. 2007, 14, 1202−1206.
(32) Fiedler, D.; Bergman, R. G.; Raymond, K. N. Angew. Chem., Int.
Ed. 2004, 43, 6748−6751.
(33) Parker, M. F. L.; Osuna, S.; Bollot, G.; Vaddypally, S.; Zdilla, M.
J.; Houk, K. N.; Schafmeister, C. E. J. Am. Chem. Soc. 2014, 136,
3817−3827.
(34) Crich, D.; Krishnamurthy, V.; Brebion, F.; Karatholuvhu, M.;
Subramanian, V.; Hutton, T. K. J. Am. Chem. Soc. 2007, 129, 10282−
10294.
(62) (a) Kries, H.; Blomberg, R.; Hilvert, D. Curr. Opin. Chem. Biol.
2013, 17, 221−228. (b) Blomberg, R.; Kries, H.; Pinkas, D. M.; Mittl,
P. R. E.; Gruetter, M. G.; Privett, H. K.; Mayo, S. L.; Hilvert, D. Nature
2013, 503, 418−421.
(63) Lavandera, I.; Kern, A.; Ferreira-Silva, B.; Glieder, A.; de
Wildeman, S.; Kroutil, W. J. Org. Chem. 2008, 73, 6003−6005.
(35) Nakai, T.; Ari-Izumi, A. Tetrahedron Lett. 1976, 17, 2335−2338.
(36) Zaim, O. Tetrahedron Lett. 1999, 40, 8059−8062.
(37) (a) Friest, J. A.; Broussy, S.; Chung, W. J.; Berkowitz, D. B.
Angew. Chem., Int. Ed. 2011, 50, 8895−8899. (b) Friest, J. A.; Maezato,
Y.; Broussy, S.; Blum, P.; Berkowitz, D. B. J. Am. Chem. Soc. 2010, 132,
5930−5931. (c) Broussy, S.; Cheloha, R. W.; Berkowitz, D. B. Org.
Lett. 2009, 11, 305−308. (d) Dey, S.; Powell, D. R.; Hu, C.; Berkowitz,
D. B. Angew. Chem., Int. Ed. 2007, 46, 7010−7014.
(38) (a) Zhao, Z.; Liu, P.; Murakami, K.; Kuzuyama, T.; Seto, H.;
Liu, H.-w. Angew. Chem., Int. Ed. 2002, 41, 4529−4532. (b) Attolini,
M.; Iacazio, G.; Peiffer, G.; Maffei, M. Tetrahedron Lett. 2002, 43,
8547−8549.
(39) Kitamura, M.; Tokunaga, M.; Noyori, R. J. Am. Chem. Soc. 1995,
117, 2931−2.
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DOI: 10.1021/jacs.5b00022
J. Am. Chem. Soc. 2015, 137, 3600−3609