Evolution of Biocatalytic and Chemocatalytic C-H Functionalization Strategy in the Synthesis of Manzacidin C

Article abstract of DOI:10.1021/acs.joc.8b00248

Because of their unique molecular architecture, the manzacidins have been the subject of intense synthetic efforts in the past two decades. Here, we describe two synthetic approaches toward manzacidin C that center on the enzymatic hydroxylation of unprotected l-leucine. This study also resulted in the discovery of novel synthetic methodologies, including a photocatalytic C-H azidation of unprotected amino acids. Additionally, we describe the use of hydroxylated l-leucine in the preparation of various densely substituted pyrrolidines.

Full text of DOI:10.1021/acs.joc.8b00248

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Article
Evolution of Biocatalytic and Chemocatalytic C–H
Functionalization Strategy in the Synthesis of Manzacidin C
Christian R Zwick, and Hans Renata
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00248 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
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Evolution of Biocatalytic and Chemocatalytic C–H Functionalization Strategy in the
Synthesis of Manzacidin C
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Christian R. Zwick, III, Hans Renata*
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458
hrenata@scripps.edu
biocatalytic
hydroxylation
Br
Me NH2
CO2H
HN
N
O
Me
N
CO2H
photocatalytic
azidation
H
Me
O
manzacidin C
L-leucine
Abstract
Due to their unique molecular architecture, the manzacidins have been the subject of
intense synthetic efforts in the past two decades. Here, we describe two synthetic approaches
towards manzacidin C that center on the enzymatic hydroxylation of unprotected L-leucine. This
study also resulted in the discovery of novel synthetic methodologies, including a photocatalytic
C–H azidation of unprotected amino acids. Additionally, we describe the use of hydroxylated L-
leucine in the preparation of various densely-substituted pyrrolidines.
Introduction
Commonly isolated from marine organisms, the bromopyrrole alkaloids1 have long
fascinated chemists due to their intricately complex structures. Three members of this family,
manzacidins A–C were isolated by Kobayashi and co-workers in 1991 from the Okinawan
sponge Hymeniacidon sp.2 Although small, the manzacidins exhibit dense structural complexity
(Figure 1A). Their core structure contains a 2,4-disubstituted bromopyrrole ester, as well as a
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tetrahydropyrimidine motif featuring a carboxylic acid and two stereocenters, including a
nitrogen-containing quaternary center.3 In 2000, Ohfune and co-workers established the first
total syntheses of manzacidin A and C, sparking what has become a thriving area in the
development of unique methodologies over the past eighteen years.4 Their landmark strategy
established lactone 5 as an advanced intermediate in thirteen steps, which can be converted to the
natural product through a highly efficient 54% two-step protocol involving formation of the
tetrahydropyrimidine motif, followed by bromopyrrole ester installation (Figure 1B). Since this
seminal work, many approaches have developed or applied new methodology towards 5,
constituting a formal synthesis of manzacidin C (the γ epimer of 5 targeted for manzacidin A).5
Although 5 appears structurally underwhelming, a literature survey suggests that approaches to
this target remain non-trivial and typically require thirteen to fifteen steps. Alternative
approaches that avoid the intermediacy of lactone 5 have also been developed, and in some
cases, have resulted in some of the most step-efficient syntheses of the manzacidins.6 Of note,
Maruoka and co-workers quickly constructed manzacidin A with high stereocontrol via an
enantioselective 1,3-dipolar cycloaddition, resulting in 17% overall yield in five steps.6a
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Figure 1. Sequential C–H functionalization approach for the synthesis of manzacidin C:
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biosynthetic inspiration and generalized synthetic strategy.
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Ohfune’s seminal publication briefly proposed that the manzacidins might have arisen
from a hydroxylated isonitrile variant of L-Leu (2, Figure 1A). Isonitrile containing natural
products are present in large abundance in marine sponges and tunicates.7 In sponges, previous
biosynthetic studies confirmed that inorganic cyanide is incorporated enzymatically to generate
the isonitrile functionality, contrasting terrestrial isonitrile formation via α-amino acids (AAs).7
Isonitrile incorporation likely occurs through a transiently-formed carbocation starting from an
alkene or tertiary alcohol. Biosynthetically, intermediate 2 may be constructed from L-Leu
through a tandem C–H dihydroxylation sequence, followed by a Ritter-type reaction with
inorganic cyanide at the γ carbon to install the isonitrile. In contrast to isonitrile functionality,
hydroxyl group incorporation via biocatalytic C–H hydroxylation is well-documented. Of note,
iron- and α−ketoglutarate-dependent dioxygenases (Fe/αKGs) are prolific in their ability to
perform site- and stereospecific hydroxylation of AAs.8 However, investigations into the
synthetic potential of these hydroxylases have remained severely overlooked.9 The ability to
manipulate and repurpose Nature’s repertoire of oxidation biocatalysts for synthetic
transformations can result in a highly enabling approach towards constructing complex
molecules.9 To demonstrate this idea with the Fe/αKGs, our laboratory chose to embark on a
chemoenzymatic synthesis of manzacidin C, employing Ohfune’s proposal as inspiration. This
full account traces the evolution of our chemoenzymatic approach towards this alkaloid and the
subsequent application of enzymatic amino acid hydroxylation in the preparation of novel
scaffolds.
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Results and Discussions
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Towards the synthesis of manzacidin C, we envisioned a C–H functionalization strategy
to access Ohfune’s hypothetical intermediate 2 (Figure 1C). Our strategy centered on efficient
C–H amination/hydroxylation transformations from the cheap and abundant starting material, L-
Leu (1). Although C–H functionalization strategies have become ubiquitous in complex
molecule synthesis, examples demonstrating remote/distal C–H functionalization are rare; even
more so for AAs (Figure S1).10 Site specific C–H functionalization largely depends on the steric
and electronic features present in a molecule.11 Selective tertiary C–H bond functionalization is
largely precedented, as electron deficient intermediates are stabilized via hyperconjugation,
yielding a high level of site selectivity. Transition metal catalyzed C–H functionalization of AAs
utilizing directing groups has recently been reported, but typically results in β functionalization
due to preferential formation of a 5-membered metallacycle intermediate.12 Hofmann-Löffler-
Freytag (HLF) chemistry has previously been developed for δ functionalization of AAs, but
displays poor regio/stereocontrol and limited substrate scope.10c Known methods for C–H
functionalization of AAs also require prudent protecting group strategies to mask highly reactive
amine/carboxylic acid functional moieties.10,12 Unlike traditional chemical methods, enzymatic
C–H functionalization of AAs displays excellent regio/chemoselectivity and is able to operate on
unprotected AAs.8 Based on this information, our approach hinges on developing chemistry to
selectively functionalize the γ and δ positions of L-Leu, ideally without the addition of protecting
groups.
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We were initially drawn to the aforementioned Fe/αKG family of enzymes due to their
known activity in the hydroxylation of AAs en route to the biogenesis of nonribosomal peptides
(NRPs) .8,13 Prior to our studies, C5 hydroxylation of L-leucine has previously been established
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in the biosynthesis of the 4-methylproline motif of the nostopeptolides and the echinocandins,
and the corresponding enzymes (LdoA and EcdK, respectively) have been biochemically
characterized.14 Due to previously reported limitations of LdoA and EcdK, we chose to
characterize a putative leucine hydroxylase called GriE, which is implicated in the biosynthesis
of griselimycin (Figure S2).15 To this end, we first heterologously overexpressed GriE in E. coli
as the C-His6-tagged protein, resulting in protein yield as high as 100mg/L. Combining GriE
with L-Leu, α-ketoglutarate, iron(II) sulfate, and L-ascorbic acid yielded 4-(OH)-L-Leu with
complete conversion and high total turnover number (TTN = 7800). In tandem with our studies,
Müller characterized GriE using a combination of in vitro studies and gene inactivation.16
Further biochemical characterization showed that GriE displays a superior kinetic profile with its
native substrate relative to previously reported Fe/αKG enzymes.8b Encouraged by these
observations, we conducted gram-scale hydroxylation of L-Leu with GriE, which following Boc
protection and facile intramolecular cyclization, provided lactone 7 in 68% yield (Scheme 1A).
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Scheme 1. C5 hydroxylation of L-Leu (1) en route to our first generation formal synthesis of
manzacidin C.
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With an efficient protocol for producing gram quantities of 7 in hand, we next targeted an
appropriate C–H amination transformation towards manzacidin C (Scheme 1B). Though
intermolecular amination or azidation of tertiary C–H bonds is well represented in the
literature,17 established procedures to effect this transformation showed no reaction on 7. This
observation is attributed to a combination of steric hindrance (axial C–H bond) and electronic
deactivation (proximal to ester oxygen).11a We next sought a more robust strategy to install the
critical C–N bond. Intramolecular nitrene insertion has seen extensive use in natural product
synthesis.18 Of note, rhodium and silver mediated strategies have become particularly powerful
approaches for effecting late-stage C–H amination. This disconnection has been applied in the
syntheses of nitrogen-containing natural products such as the welwitindolinones, tetrodotoxin,
pactamycin, and most notably manzacidin A and C by Du Bois.18,19 Mechanistic studies indicate
that a carbamate/sulfamate is first converted to an iminoiodinane (formally a nitrene) by the
hypervalent iodine reagent (e.g., phenyliododiacetate (PIDA) or iodosobenzene (PhIO)).
Subsequently, the metal catalyst (Ag(I) or Rh(II)) decomposes the iminoiodinane, yielding an
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electrophilic metal nitrenoid, which performs insertion at proximal/electronically rich C–H
bonds.18 Our revised strategy involved installation of a carbamate moiety, which would
participate in the proposed C–H amination (Scheme 1C). Towards carbamate 8, we first
converted lactone 7 to the Weinreb amide using a combination of trimethylaluminum and N,O-
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dimethylhydroxylamine
hydrochloride,
followed
by
carbamate
formation
with
trichloroacetylisocyanate, resulting in 76% two-step yield on gram scale.19c At this stage, the
critical C–H insertion could be tested. While catalytic rhodium(II) acetate dimer (Rh2(OAc)4)
gave no reaction, the more active catalyst Rh2esp2 yielded the desired product in 53% yield.20
Previously reported conditions utilizing silver(I) triflate, bathophenanthroline, and PIDA in
MeCN at 82 °C yielded oxazolidinone 9 in 65% (77% brsm).19a A two-step procedure involving
Boc protection of the oxazolidinone, followed by methanolysis and lactonization yielded 5 in
50% yield over two steps. This sequence constitutes a nine-step formal synthesis of manzacidin
C (11% total yield), exhibiting complete stereocontrol through sequential C–H
functionalizations.
Scheme 2. Failed attempts to convert 4,5-dihydroxyleucine (11) to a key intermediate towards
manzacidin C.
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A. Failed functionalization of 4,5-(OH)2-Leu
Gox, αKG,
50 mM kPi
(pH = 7.0)
GriE, αKG,
50 mM kPi
(pH = 7.0)
OH
Me
CO2H
Me
HO
CO2H
CO2H
HO
enzymatic
-hydroxylation
enzymatic
-hydroxylation
Me NH2
Me NH2
Me NH2
11
γ
δ
1
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BzCl,
NaHCO3
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O
OH
Me
Lewis acids,
nucleophile
O
CO2H
NHBz
Me
Nu NHBz
BzO
12
B. Discovery of azidolysis of lactone 13
1. TFAA
2. Sc(OTf)3,
TMSCN
O
SnCl4, TMSN3,
MeNO2
R2
R3
Me
N3
OH
Me
CO2H
NHBz
R1
O
NHBz
R1
R3
R2
Me
Me
82%
Shenvi,
2013
NC
13
14
Although our formal synthesis has improved step count relative to those previously
reported, it lacks optimal synthetic ideality.21 We identified that our critical C–N bond
disconnection necessitated several functional group interconversions (FGIs) and protecting group
manipulations (PGMs), leading to the poor ideality and step count. For this reason, a more
efficient strategy to access 5 was sought. Concurrent to our effort in developing a
chemoenzymatic synthesis of manzacidin C, we were also able to establish the substrate scope of
the enzymatic hydroxylation.9a A key observation derived from the substrate profiling of GriE is
that substitution at the γ position of the amino acid substrate is tolerated. In particular, 4-(OH)-
Leu22 could be oxidized to the corresponding dihydroxy amino acid (11) in moderate TTN
(Scheme 2A). Elaboration of this compound to lactone 5 was next investigated. In particular,
displacement of the tertiary alcohol with an appropriate nitrogenous nucleophile would closely
mimic our postulated biosynthesis of the manzacidins. Inspired by the work of Shenvi and co-
workers (Scheme 2B), as well as related work on Lewis/Brønsted Acid-mediated opening of
butyrolactones, we reasoned prudent combination of Lewis Acid and nucleophile may yield our
desired C–N bond.23 This process likely proceeds through the intermediacy of a contact ion pair,
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dictating stereochemical inversion during isonitrile formation. If stereoinvertive, this would
allow the synthesis of manzacidin A, which is epimeric at the γ position with respect to
manzacidin C (Figure 1A). Unfortunately, lactone 12 showed no reaction under Shenvi’s
conditions. After screening various Lewis Acids in combination with azidotrimethyl silane
(TMSN3), we discovered combination of SnCl4 in a 1:1 mixture of TMSN3:MeNO2 resulted in
epimerization at the γ carbon (no other conditions showing any reaction). While disheartening,
this result suggested that a carbocation was transiently formed under the reaction conditions. If
this were the case, generating a more persistent/electron rich carbocation may yield the desired
reactivity. To test this hypothesis, lactone 13 was subjected to the reaction conditions (Scheme
2B, see Experimental Procedures for preparation of 13), which resulted in the formation of the
desired azidocarboxylic acid 14. Subsequent optimization of the reaction conditions improved
the yield of 14 to 82% yield (see Table S1). Attempting the azidolysis reaction in other solvents
besides MeNO2 resulted in no reaction (see Table S1). Strict MeNO2 requirement with SnCl4 has
similarly been reported for intramolecular Freidel-Crafts reactions with cyclopropylesters,
wherein a carbocation intermediate was also proposed as a transient intermediate.24 Similarly,
hydrobromic acid has been shown to open α-tertiary lactones yielding a tertiary bromocarboxylic
acid, also likely proceeding via a carbocation intermediate.25 Interestingly, other azide sources
such as sodium azide or tetrabutylammonium azide showed no reaction, suggesting TMSN3 is
required for the reaction to proceed. Though fruitless towards the synthesis of manzacidin A, this
chemistry is currently under further synthetic and mechanistic investigations.
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Scheme 3. Second generation formal synthesis of manzacidin C utilizing C–H azidation and
biocatalytic C–H hydroxylation.
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Britton, 2017:
TBADT,
NFSI,
hν (365 nm)
accepts longer
chain
sensitive to
sterics
O
O
F
Me
CO2H
Me
Me
OH
OH
tolerates
substitution
H
>85%
conversion
Me NH2
Me NH2
Me NH2
A. Discovery of C–H azidation of 1 and attempt at converting 16 to 5
HO
b. GriE, O2,
a. 16, TBADT,
N3
N3
αKG, Fe2+
365 nm hν
Me
CO2H
Me
CO2H
CO2H
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>95% conversion
130 mg scale
49%
500 mg scale
Me NH2
1
Me NH2
16
Me NH2
17
F3C
SO2N3
c. Boc2O
15
58% over
2 steps
CF3
O
NHBoc
O
d. H2, PtO2
HN
NHBoc
O
Me
N3
O
Me
HO
Me
55%
19
O
NH2
NHBoc
18
pseudo-boat
B. Final route for the formal synthesis of manzacidin C from L-leucine
c. H2, Pt/C,
O
N3
NH2
K2CO3;
Boc2O;
CO2H
CO2H
Me
O
HO
HO
BocHN
41% over
2 steps
HCl
Me NH2
17
Me NH2
20
NHBoc
5
Based on the above foray, we realized the strategic benefit of using a pre-functionalized
substrate for the enzymatic hydroxylation in improving the step efficiency of our synthesis.
Specifically, early installation of the γ C–N bond in the synthesis would avoid subsequent
concession steps, including the tethering strategy for a stereospecific intramolecular C–H
amination. The proposed strategy would also dovetail nicely with GriE-mediated δ
hydroxylation, which based on our substrate scope model accepts substrates with additional
substitution at the γ position. This strategy is not without its challenge, as methods to effect
direct C–H amination on unprotected AAs are not well-established. However, Britton’s recent
work on the γ selective C–H fluorination of unprotected L-Leu with tetrabutylammonium
decatungstate (TBADT) as photocatalyst served as an inspiration to develop an analogous
protocol for amination.26 In the proposed catalytic cycle,27 TBADT selectively performs
hydrogen atom abstraction on the substrate to generate a persistent tertiary carbon centered
radical. Subsequent trapping of this species by N-fluorobenezesulfonimide (NFSI) results in C–F
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bond formation at the γ position. We reasoned that a similar electrophilic azide source may prove
amenable to form our desired C–N bond.10b,17a,b Indeed, when L-Leu•TFA was stirred with 5
mol% TBADT and 3 equiv. of azide 15, azidoleucine 16 was obtained; albeit as a 1:2.5 SM:PD
mixture, which was separated by preparative HPLC (Scheme 3A). This reaction proved scalable,
resulting in 49% yield using 500 mg of L-Leu•TFA (SM was not recovered). This is the first
example of TBADT mediated azidation, as well as the first C–H azidation protocol on
unprotected AAs. Due to the unique reactivity of the catalytic manifold, extension of this
photoreaction is currently under further investigation in our group. Biocatalytic hydroxylation of
16 with GriE yielded difunctionalized amino acid 17 in >95% conversion on 130 mg scale. This
sequence is remarkable, as two sequential C–H functionalizations on unprotected L-Leu could be
effected. At this stage, 17 was Boc protected and lactonized to 18 in 58% yield over two steps.
All that remained was a simple reduction and Boc protection to finish our synthesis. To our
surprise, submitting 18 to reductive conditions (H2/PtO2) resulted in complete conversion to
lactam 19 in 55% yield. A quick literature search revealed that the same by-product was
previously obtained during reduction of a structurally similar nitrolactone.28 Presumably, after
azide reduction the molecule can adopt a pseudo boat conformation, which allows the tertiary
amine to participate in an intramolecular addition-elimination with the lactone carbonyl to afford
19. This pathway also occurred in the presence of triphenylphosphine (PPh3), presumably
through a Staudinger reaction. No desired product was ever formed, even when the reaction was
run in the presence of Boc2O or Boc-ON to capture the transiently formed amine after reduction.
Though 19 was considered to complete the synthesis of manzacidin C via Boc protection and
methanolysis, it would only improve our previous synthesis by one step. Nevertheless, the facile
lactamization provided a key insight to choreograph an alternative sequence to access 5 from 17
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(Scheme 3B). We reasoned that under basic pH, the carboxylic acid would exist as the
carboxylate, which would have a much-reduced propensity to undergo lactamization during the
azide reduction and Boc protection sequence. Thus, a one-pot procedure was developed wherein
17 was first reduced, the resulting free amines (20) subsequently Boc protected, and
alcohol/carboxylic acid motifs lactonized to provide 5 in 41% yield over two steps. This
constitutes a five-step formal synthesis of manzacidin C in 11% overall yield. Although
displaying equivalent overall yield to our first generation synthesis, it is remarkably shorter and
displays significantly improved ideality over the first three transformations.
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Scheme 4. Synthetic access to complex pyrrolidine scaffolds starting from lactone 7.
During the course of our studies, we also sought to demonstrate the utility of GriE in the
synthesis of other complex and biologically relevant molecules (Scheme 4). Since hydroxylated
AAs are prevalent in nonribosomal peptides (NRPs), 7 was submitted to a peptide coupling by
simple heating with L-glycine ethyl ester (21) to provide dipeptide 22 in excellent yield.29
Standard peptide coupling requires the use of carboxylic acid activating agents, whereas lactones
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represent a form of protected and pre-activated carboxylic acids, obviating the use of external
coupling reagents. Lactone 7 was converted to the piperidyl amide, followed by Parikh-Doering
oxidation to yield hemiaminal 23. This functionalized proline variant features increased
oxidation state at the δ position, serving as a handle for further derivatization. Synthetic
manipulation of similarly oxidized pyrrolidines—albeit lacking methylation at the δ position—
has recently been reported in the literature.30 To demonstrate this, 23 was reacted with
allyltrimethylsilane (24) in the presence of BF3•Et2O to yield 25 in 49% yield as a 4:1 mixture of
diastereomers.31 Carbapenam β-lactams represent structurally intricate scaffolds en route to
carbapenem antibiotics.13b Since hemiaminal 23 exists in an equilibrium with the free aldehyde,
Horner-Wadsworth-Emmons (HWE) reaction yielded a mixture of the homologated pyrrolidine
(S-03) and its open chain tautomer (S-04) (Figure S3).30a Submitting the mixture directly to
trifluoroacetic acid resulted in complete conversion to the corresponding amino acid (S-05) in
63% yield over two steps. Dehydration with Mukaiyama reagent (2-chloro-1-methylpyridinium
iodide) afforded γ-methylated carbapenam 27 in 41% yield as a 5:1 mixture of diastereomers.32
This sequence resulted in 13% yield over five steps from 7.
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Scheme 5. Chemoenzymatic synthesis of N-protected and free (2S,4R)-4MePro.
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Due to the ubiquity of (2S,4R)-4-MePro (28) in a variety of biologically interesting
NRPs, a scalable and efficient synthesis of this motif is highly desirable.13-15 A previous
approach to 28 requires an alcohol directing group and uses expensive Crabtree’s catalyst to
perform asymmetric reduction of an exocyclic olefin, yielding the desired stereochemistry with
40:1 dr.33 Towards (2S,4R)-4-MePro, 7 was converted to the Weinreb amide as previously
described (vide supra). The resulting alcohol was then mesylated and cyclized in one-pot upon
addition of KOtBu. Hydrolysis of the Weinreb amide with LiOH provided Boc-(2S,4R)-4-MePro
(29) in 44% over three steps on 500 mg scale and 30% overall yield from L-Leu (Scheme 5A).
Our synthesis compares favorably to the hydrogenation route as it proceeds with complete
stereocontrol and avoids directing groups and expensive catalysts. An observation during our
substrate scope examination of GriE spurred further work in this area. It was observed that
hydroxylation of γ-Me-L-Leu with GriE resulted in minor amounts of over oxidation to an
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aldehyde, which is present in solution as the cyclic imine. This was similarly observed with L-
Leu as well as other γ-substituted L-Leu variants with increased enzyme loading. Drawing
inspiration from (2S,4R)-4-MePro’s biosynthesis (see Supporting Information), we reasoned that
conducting the reaction with sufficiently high GriE concentration, followed by addition of a mild
reductant could yield γ-substituted L-Pro derivatives in a single step (Scheme 5B).34 To our
delight, this chemoenzymatic cascade could be performed on L-Leu and its derivatives—
commercially available or synthesized from L-Leu in one step utilizing TBADT mediated
photochemistry or Gox hydroxylation—, resulting in five unique proline derivatives in one step.
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Conclusion
During the course of this work we demonstrated the unique advantage of combining
enzymatic and traditional chemical methods in complex molecule synthesis.8b Our initial
attempts to emulate the biogenesis of manzacidin C in the laboratory resulted in the
characterization of a leucine hydroxylase, GriE, that can be used for preparative scale
hydroxylation of a wide range of AAs. Additionally, our efforts to address the construction of the
quaternary center of the target molecule yielded interesting new methodologies for nucleophilic
opening of lactones with azides and photocatalytic C–H azidation of unprotected AAs. These
discoveries culminated in an efficient formal synthesis of manzacidin C in just five steps. We
further demonstrated the synthetic utility of amino acid oxidation with GriE by developing a
chemoenzymatic approach to a variety of structurally and biologically interesting molecules,
including novel proline analogs. Further applications of this synthetic paradigm35 in the
preparation of bioactive natural products are on-going and will be reported in due course.
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Experimental Procedures
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Compounds 5, 6, 7, 10, 12, 15, 16, 17, 20, 22, 28, 30, 31, 32, and 33 were previously synthesized
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and characterized.9a
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tert-butyl ((2S,4R)-5-(carbamoyloxy)-1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-
yl)carbamate (8)
A flame-dried 100 mL round bottom (w/ stir bar) was sequentially charged with N,O-
dimethylhydroxylamine hydrochloride (1.21 g, 12.44 mmol, 3.0 eq) and 12.4 mL anhydrous
THF. The suspension was stirred under Ar and cooled to 0 °C using an ice bath. AlMe3 (2 M in
PhMe, 6.2 mL, 12.44 mmol, 3.0 eq) was added via syringe pump over 30 min. The resulting
solution was stirred for 1h at 23 °C, then cooled to –30 °C using a dry ice/acetone bath. Lactone
7 (0.951 g, 4.14 mmol, 1.0 eq, 0.5 M in THF) was added to a dry scintillation vial and dissolved
in anhydrous THF (8.3 mL). The resulting solution was added dropwise over 10 min to the round
bottom at –30 °C. After 30 min, the reaction was then quenched at –30 °C with 20 mL 10%
(w/w) aq. Rochelle’s salt (20 mL) and Et2O (20 mL). The resulting mixture was allowed to stir at
23 °C for 30 min, diluted with H2O and filtered over celite. The aq. layer was then extracted with
Et2O (3 x 20 mL). The organics were washed with sat. aq. NaCl (50 mL), dried over MgSO4,
filtered, and concentrated by air stream. The resulting oil was immediately transferred to a 250
mL round bottom flask, placed under Ar, and diluted with anhydrous DCM (83 mL). This
solution was cooled to –40 °C, and trichloroacetyl isocyanate (740 µL, 6.22 mmol, 1.2 eq) was
added dropwise with stirring. The reaction was stirred for 30 min at this temperature, then
warmed to 23 °C and concentrated in vacuo. The resulting oil was diluted with MeOH (56 mL)
and K2CO3 (860 mg, 6.22 mmol, 1.5 eq) was added. The resulting slurry was stirred for 30 min,
filtered over celite, and concentrated in vacuo. The crude oil was purified by silica gel
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chromatography via gradient elution (1:1 EtOAc:hexanes to 100% EtOAc), affording 8 (1.11 g,
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76% yield) as a yellow foam. H NMR (400 MHz, CDCl3): δ 5.12 (d, J = 9.8 Hz, 1H), 4.92 (bs,
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1H), 4.07 (dd, J = 10.6, 4.8 Hz, 1H), 3.85 (dd, J = 10.6, 7.4 Hz, 1H), 3.79 (s, 3H), 3.19 (s, 3H),
2.05 – 1.94 (m, 1H), 1.71 (ddd, J = 13.3, 8.8, 4.2 Hz, 1H), 1.49 – 1.42 (m, 1H), 1.42 (s, 9H), 0.96
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(d, J = 6.9 Hz, 3H); C NMR (101 MHz, CDCl3): δ 173.6, 157.1, 155.3, 79.9, 69.8, 61.8, 48.9,
23
38.0, 32.2, 29.5, 28.5, 17.8; Rf = 0.30 (2:8 EtOAc:hexanes); [α]D = 1.64º (c = 0.3, CHCl3);
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H27N3O6H+ 334.1978; Found 334.1977.
tert-butyl ((S)-1-(methoxy(methyl)amino)-3-((S)-4-methyl-2-oxooxazolidin-4-yl)-1-
oxopropan-2-yl)carbamate (9)
Two 20 mL Biotage® microwave vials were flame dried (w/ stir bars). To each vial was added
225 µL of 1 M stock solution of 8 in DCM. The vials were placed on high vacuum for 30 min,
resulting in immediate formation of a yellow foam. After refilling with Ar, the vials were moved
to a glovebox, wherein AgOTf (35 mg, 0.18 mmol, 0.25 eq), bathophenanthrolene (0.182 g, 0.55
mmol, 0.5 eq), and anhydrous/degassed MeCN (4 mL) was added to each vial. The vials were
covered with foil and stirred for 30 min. Subsequently, 4 mL MeCN and PIDA (0.182 g, 0.55
mmols, 1.0 eq) were added to each vial. Each vial was sealed, wrapped in foil, removed from the
glovebox, and heated to 82 °C (with stirring) in an oil bath for 24 h. The reactions were cooled to
23 °C, combined, filtered over a plug of silica with EtOAc, and concentrated in vacuo. The
resulting crude material was purified by silica gel chromatography via gradient elution (1:1
EtOAc:hexanes to EtOAc), affording 8 (22 mg, 12% yield) and 9 (0.117 g, 65%, white solid). 1H
NMR (400 MHz, CDCl3): δ 6.11 (s, 1H), 5.46 (d, J = 9.0 Hz, 1H), 4.81 (t, J = 9.7 Hz, 1H), 4.11
(d, J = 8.4 Hz, 1H), 4.03 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H), 3.21 (s, 3H), 2.03 (dd, J = 14.0, 3.0
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Hz, 1H), 1.69 (dd, J = 13.9, 10.5 Hz, 1H), 1.44 (s, 9H), 1.41 (s, 3H); 13C NMR (101 MHz,
CDCl3): δ 172.0, 158.3, 155.89, 80.8, 62.02, 57.0, 48.0, 42.1, 32.4, 28.5, 25.6; Rf = 0.20 (2:8
EtOAc:hexanes). [α]D20 = –28.5º (c = 0.1, CHCl3); mp = 94 °C; HRMS (ESI-TOF) m/z: [M+H]+
- [CO2 + CH2C(CH3)2] Calcd for C9H17N3O4H+ 232.1297; Found 232.1287.
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di-tert-butyl ((3S,5S)-5-methyl-2-oxotetrahydro-2H-pyran-3,5-diyl)dicarbamate (5)
Carbamate 9 (0.104 g, 0.31 mmol, 1.0 eq) was added to a scintillation vial (w/ stir bar) and
dissolved in 3.1 mL THF (0.1M) with stirring. The solution was subsequently treated with TEA
(174 µL, 1.25 mmol, 4.0 eq), DMAP (76 mg, 0.62 mmol, 2.0 eq), and Boc2O (0.273 g, 1.25
mmol, 4.0 eq). After stirring 1.5 h, the reaction was quenched with sat. aq. NH4Cl (3.0 mL),
extracted with EtOAc (3 x 10 mL), and washed with sat. aq. NaCl. The organics were dried over
MgSO4, filtered, and concentrated in vacuo. The resulting material was diluted with 6.3 mL
MeOH (0.05 M), cooled using an ice bath (0 °C), and K2CO3 (86 mg, 0.62 mmol, 2.0 eq) was
added. The resulting slurry was warmed to 23 °C and stirred for 12 h. After concentrating in
vacuo, the mixture was dissolved in 10 mL H2O and 10 mL EtOAc. The pH of this mixture was
adjusted to 3 using AcOH. The aq. layer was extracted with EtOAc (3 x 10 mL) and
concentrated in vacuo. The resulting oil was diluted with 10 mL PhMe. MgSO4 (~0.5 g) was then
added, and the resulting slurry was stirred vigorously with heating for 2 h at 70 °C using an oil
bath. The slurry was cooled to 23 °C, filtered, and concentrated in vacuo. The resulting crude
material was purified by silica gel chromatography via gradient elution (dry loaded) (2:98
1
MeOH:DCM to 5:95 MeOH:DCM), affording 5 (54 mg, 50% yield) as a white solid. H NMR
spectrum matches previous report.9a 1H NMR (400 MHz, CDCl3): δ. 5.30 (bs, 1H), 4.71 (bs, 1H),
4.57 (bs, 2H), 4.25 (bs, 1H), 2.75 (bs, 1H), 1.66 (t, J = 13.0 Hz, 1H), 1.46 (s, 9H), 1.44 (s, 9H),
1.39 (s, 3H).
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(S)-N-(5,5-dimethyl-2-oxotetrahydrofuran-3-yl)benzamide (13)
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Following previously reported procedure,9a E. coli expressing Gox were grown on 400 mL scale.
After harvest, the cells were resuspended to OD600 = 45 in 50 mM kPi buffer (pH 7.0). Cells were
disrupted by sonication (3x1 min, 50% duty cycle) and pelleted by centrifugation at 4,000 rpm
for 15 min at 4 ºC. A 250 mL Erlenmeyer flask was charged with L-leucine (328 mg, 2.5 mmol,
1.0 equiv), L-ascorbic acid (440 mg, 2.5 mmol, 1.0 equiv), α-ketoglutaric acid (disodium salt
dihydrate, 1.69 g, 7.5 mmol, 3.0 equiv), and FeSO4 (heptahydrate, 28 mg, 0.1 mmol, 0.04 equiv).
After addition of clarified Gox lysate (50 mL), the flask was shaken at 20 ºC for 7 h at 250 rpm.
Reaction was quenched by addition of 1 M HCl (20 mL) and centrifuged at 4,000 rpm for 15 min
at 4 ºC. The supernatant was collected and concentrated to dryness and was used without further
purification. The crude material was resuspended in sat. aq. NaHCO3 (~ 75 mL). To this
suspension was added a solution of BzCl (1.16 mL, 10 mmol, 4.0 equiv) in 75 mL DCM and the
mixture was stirred vigorously at room temperature overnight. The pH of the mixture was
adjusted to 2–3 and the layers were separated. The aqueous phase was extracted with DCM (2 x
50 mL), and the combined organic layers were washed with sat. aq. NaCl (50 mL), dried over
MgSO4, and concentrated in vacuo. The resulting crude material was purified by silica gel
chromatography via gradient elution (1:4 EtOAc:hexanes to 1:1 EtOAc:hexanes) to afford
lactone 13 (269 mg, 46% yield over 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3): δ 7.80 (d,
J = 7.2, 2H), 7.55 (d, J = 7.3 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 6.63 (s, 1H), 4.92 (ddd, J = 11.7,
8.7, 5.4 Hz, 1H), 2.90 (dd, J = 12.6, 8.6 Hz, 1H), 2.08 – 1.97 (t, J = 12.6 1H), 1.56 (s, 6H); 13C
NMR (101 MHz, CDCl3): δ 175.4, 167.7, 133.1, 132.2, 128.7, 127.2, 83.4, 50.8, 42.5, 29.1, 27.1; Rf =
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0.40 (1:1 EtOAc:hexanes); [α]D
= 47.0º (c = 0.3, CHCl3); mp = 121 °C; HRMS (ESI-TOF) m/z:
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[M+H]+ Calcd for C13H15NO3H+ 234.1130; Found 234.1121.
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(S)-4-azido-2-benzamido-4-methylpentanoic acid (14)
To a flame-dried 10 mL round bottom flask (w/ stir bar) under Ar, was added 13 (30 mg, 0.13
mmol, 1.0 eq). The flask was charged with 220 µL MeNO2 and 220 µL TMSN3 (both dried over
4Å molecular sieves prior to use). The resulting solution was treated dropwise with anhydrous
SnCl4 (1 M in heptane) (424 µL, 0.42 mmol, 3.3 eq) at 0 ºC. The reaction was stirred at 0 °C for
1 h, immediately warmed to 23 °C, and stirred at this temperature for 48 h. The reaction was
diluted with 1 mL EtOAc and quenched with 1 mL H2O with vigorous stirring. The resulting
solution was adjusted to pH = 1 using 1 N aq. HCl. The aq. layer was extracted with EtOAc (3 x
5 mL) and the combined organics were washed with sat. aq. NaCl, dried over MgSO4, filtered,
and concentrated in vacuo. The resulting crude oil was then purified by silica gel
chromatography (1:1 EtOAc:hexanes, then gradient elution 1:99 MeOH:DCM 1% AcOH to 3:97
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MeOH:DCM 1% AcOH), affording 14 (29 mg, 82% yield) as a yellow oil. H NMR (400 MHz,
CDCl3): δ 7.86 – 7.78 (m, 2H), 7.59 – 7.50 (m, 1H), 7.46 (dd, J = 8.4, 6.9 Hz, 2H), 7.27 (s, 1H),
13
4.75 (ddd, J = 9.9, 6.2, 4.2 Hz, 1H), 2.20 – 1.99 (m, 2H), 1.43 (d, J = 19.3 Hz, 6H); C NMR
(101 MHz, CDCl3): δ 176.7, 175.3, 168.2, 133.1, 132.4, 128.9, 127.3, 60.9, 51.1, 41.6, 27.2,
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25.3, 20.8; Rf = 0.15 (5:95 MeOH:DCM 1% AcOH); [α]D = – 11.8º (c = 0.3, CHCl3); HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C13H16N4O3H+ 277.1301; Found 277.1300.
tert-butyl ((3S,5S)-5-azido-5-methyl-2-oxotetrahydro-2H-pyran-3-yl)carbamate (18)
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A scintillation vial was charged with 4-azidoleucine (TFA salt, 22 mg, 0.08 mmol, 1.0 equiv), L-
ascorbic acid (7.0 mg, 0.04 mmol, 0.5 equiv), α-ketoglutaric acid (disodium salt dihydrate, 18
mg, 0.08 mmol, 1.0 equiv), followed by 4.0 mL of 50 mM kPi buffer (pH 7.5). After addition of
20 µL of 200 mM aq. FeSO4 solution (4.0 µmol, 0.05 equiv), the reaction was started by the
addition of GriE stock solution (final concentration = 0.015 mM, 0.00075 equiv). The mixture
was shaken at 20 ºC, 250 rpm. After 2 h, L-ascorbic acid (7.0 mg, 0.04 mmol, 0.5 equiv), α-
ketoglutaric acid (disodium salt dihydrate, 18 mg, 0.08 mmol, 1.0 equiv), 20 µL of 200 mM aq.
FeSO4 solution (4.0 µmol, 0.05 equiv), and GriE stock solution ( 0.00075 equiv) were added and
the mixture was shaken further for 3 h. The reaction was acidified with 1 M HCl (1 mL) and
centrifuged at 15,000 rpm for 15 min. The supernatant was collected and lyophilized. The
resulting off-white powder was treated with 2 M NaOH until pH ~ 9.0 and H2O was added to
adjust the volume to ~ 2.5 mL. To this suspension was added a solution of Boc2O (61 mg, 0.28
mmol, 3.5 equiv) in 2.5 mL EtOH and the mixture was stirred at room temperature overnight.
The pH of the solution was adjusted to 2–3 and the mixture was concentrated in vacuo to remove
EtOH. To the resulting brown slurry was then added DCM (4 mL) and the mixture was stirred
overnight at room temperature. The layers were separated, the aqueous phase was extracted with
DCM (2 x 2 mL), and the combined organic layers were washed with sat. aq. NaCl (5 mL), dried
over MgSO4, and concentrated in vacuo. Purification by silica gel chromatography (1:5 to 1:1
EtOAc:hexanes) afforded lactone 18 (12.5 mg, 58% yield over 2 steps) as a white solid. 1H NMR
(400 MHz, CDCl3): δ 5.30 (bs, 1H), 4.31 (dt, J = 12.8, 6.7 Hz, 1H), 4.20 (s, 2H), 2.50 (ddt, J = 13.7,
6.9, 1.2 Hz, 1H), 1.98 (t, J = 13.0 Hz, 1H), 1.45 (d, 12H); 13C NMR (101 MHz, CDCl3): δ 170.3,
155.5, 80.8, 74.2, 58.7, 47.8, 38.6, 28.4, 22.9; Rf = 0.53 (1:1 EtOAc:hexanes). [α]D20 = 14.0º (c =
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1.1, CHCl3); mp = 94 °C; HRMS (ESI-TOF) m/z: [M+H]+ - [CO2 + CH2C(CH3)2] Calcd for
C6H10N4O2H+ 171.0882; Found 171.0879.
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tert-butyl ((3S,5S)-5-(hydroxymethyl)-5-methyl-2-oxopyrrolidin-3-yl)carbamate (19)
To a scintillation vial (w/ stir bar) was added PtO2 (1.6 mg, 0.007 mmol, 0.2 eq), 18 (9.4 mg,
0.035 mmol, 1.0 eq) and 500 µL MeOH (0.07 M). The solution was degassed by bubbling with
H2 for 15 min (during this time solution went from brown to black). The reaction was left under
an atmosphere of H2 and stirred for 3 h. The slurry was filtered over celite, and concentrated in
vacuo. The resulting crude material was purified by silica gel chromatography using gradient
elution (5:95 MeOH:DCM to 10:90 MeOH:DCM), affording 19 (4.7 mg, 55% yield) as a white
film. 1H NMR spectrum matches that reported in the literature.28 1H NMR (400 MHz, CDCl3): δ
7.01 (s, 1H), 5.53 (d, J = 7.0 Hz, 1H), 4.39 (d, J = 9.0 Hz, 1H), 3.52 (d, J = 11.5 Hz, 1H), 3.39
(d, J = 11.5 Hz, 1H), 2.27 (t, J = 11.4 Hz, 1H), 1.98 (dd, J = 13.1, 8.2 Hz, 1H), 1.44 (s, 9H), 1.25
(s, 3H).
tert-butyl (3R,5S)-2-hydroxy-3-methyl-5-(piperidine-1-carbonyl)pyrrolidine-1-carboxylate
(23)
To a flame-dried 50 mL round bottom flask (w/ stir bar) under Ar was added piperidine (204 µL,
2.06 mmol, 1.2 eq) and 4.1 mL anhydrous PhMe. The solution was cooled to 0 °C using an ice
bath, and AlMe3 (2 M solution in PhMe, 1.03 mL, 2.06 mmol, 1.2 eq) was added dropwise. The
reaction was immediately warmed to 23 °C and stirred for 45 min. Lactone 7 (0.394 g, 1.72
mmol, 1.0 eq) was added to a flame-dried scintillation vial under Ar and dissolved in 6.9 mL
anhydrous PhMe (0.25M). The resulting solution was added to the round bottom flask dropwise
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at 23 °C. The reaction was stirred for 2 hours and quenched with 30% (w/w) aq. Rochelle’s salt
(20 mL), stirred for 30 min, then transferred to a separatory funnel. Following dilution with
DCM (20 mL), the aq. layer was subsequently extracted with DCM (3 x 10 mL). The organics
were washed with sat. aq. NaCl (20 mL), dried with MgSO4, filtered, and concentrated in vacuo.
The crude alcohol was dissolved in anhydrous DCM (17 mL, 0.1M). The resulting solution was
subsequently treated with DMSO (1.2 mL, 17.2 mmol, 10.0 eq), TEA (1.2 mL, 8.62 mmol, 5.0
eq), and cooled to 0 °C using an ice bath. SO3•pyr (0.823 g, 5.17 mmol, 3.0 eq) was added and
the reaction was allowed to warm slowly to 23 °C over 16 h. The reaction was quenched with
H2O (1 mL) and the aq. layer was extracted with DCM (3 x 10 mL). The combined organics
were washed with sat. aq. NaCl (3 x 5 mL), dried over MgSO4, filtered, and concentrated in
vacuo. The resulting crude material was purified by silica gel chromatography using gradient
elution (10:90 EtOAc:hexanes to 50:50 EtOAc:hexanes), affording 23 (0.269 g, 50% over two
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steps, isolated as a mixture diastereomers and rotamers) as a colorless viscous oil. H NMR (400
MHz, CDCl3): δ 5.52 (d, J = 5.7 Hz, 1.4H), 5.38 (d, J = 5.5 Hz, 1H), 5.29 (s, 1.7H), 5.19 (d, J =
1.7 Hz, 1.6H), 5.06 (d, J = 1.6 Hz, 2.2H), 4.88 (dd, J = 9.0, 5.3 Hz, 2.5H), 4.78 – 4.67 (m, 2.8H),
4.64 – 4.56 (m, 1.7H), 3.84 – 3.75 (m, 2H), 3.66 (ddt, J = 29.7, 10.5, 6.0 Hz, 7H), 3.57 – 3.36
(m, 18H), 3.28 (td, J = 9.1, 8.7, 4.4 Hz, 3.3H), 2.49 – 2.23 (m, 6.8H), 2.15 – 1.93 (m, 6H), 1.88 –
1.70 (m, 9H), 1.71 – 1.53 (m, 30H), 1.47 (s, 31H), 1.42 (d, J = 3.4 Hz, 29H), 1.05 – 0.95 (m,
13
17H); C NMR (101 MHz, CDCl3): δ 172.4, 172.0, 169.8, 169.6, 169.4, 154.5, 154.1, 153.7,
125.4, 125.1, 115.2, 88.2, 87.9, 83.4, 83.1, 80.9, 80.7, 80.6, 80.4, 80.3, 80.2, 56.8, 56.7, 56.6,
56.2, 53.6, 47.0, 46.6, 46.4, 44.0, 43.7, 43.4, 42.2, 40.8, 39.0, 36.3, 35.5, 35.3, 35.1, 34.7, 31.1,
28.7, 28.6, 28.5, 28.5, 28.5, 26.7, 26.5, 26.4, 25.8, 25.6, 24.7, 24.6, 18.4, 18.1, 13.5, 12.8, 12.7;
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Rf = 0.30 (1:1 EtOAc:hexanes); [α]D = – 70.5º (c = 0.4, CHCl3); HRMS (ESI-TOF) m/z:
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[M+H]+ - [CO2 + CH2C(CH3)2] - [OH] Calcd for C11H18N2OH+ 195.1497; Found 195.1497.
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tert-butyl (2S,3R,5S)-2-allyl-3-methyl-5-(piperidine-1-carbonyl)pyrrolidine-1-carboxylate
(25)
To a flame-dried 10 mL round bottom flask under Ar (w/ stir bar) was added 23 (28 mg, 0.090
mmol, 1.0 eq) followed by 900 µL anhydrous DCM (0.1M). The reaction was cooled to –78 °C
using a dry ice acetone bath, at which point allyltrimethylsilane (70 µL, 0.45 mmol, 5.0 eq) and
BF3•Et2O (17 µL, 0.14 mmol, 1.5 eq) were sequentially added dropwise to the solution. After 1 h
the reaction was quenched at – 78 °C with sat. aq. NaHCO3 (1 mL). The resulting solution was
warmed to 23 °C and diluted with 5 mL DCM. The aq. layer was extracted with DCM (3 x 5
mL). The organics were then washed with sat. aq. NaCl, dried over MgSO4, filtered, and
concentrated in vacuo. The resulting crude material was purified by silica gel chromatography
using gradient elution (3:7 EtOAc:hexanes to 4:6 EtOAc:hexanes), affording 25 (14.9 mg, d.r. =
1
3.6:1.0, 49%, mixture of rotamers) as a colorless oil. Characterization data for β-epimer: H
NMR (400 MHz, CDCl3): δ 5.88 (dddd, J = 16.6, 10.2, 8.2, 6.2 Hz, 2H), 5.07 (d, J = 17.1 Hz,
2H), 5.01 (d, J = 10.2 Hz, 2H), 4.79 – 4.70 (m, 1H), 4.62 (t, J = 7.8 Hz, 1H), 3.76 – 3.61 (m,
2H), 3.58 – 3.40 (m, 6H), 2.80 (bs, 1H), 2.69 (bs, 1H), 2.45 – 2.27 (m, 2H), 2.19 (s, 2H), 1.98
(dt, J = 13.1, 6.8 Hz, 2H), 1.75 (ddd, J = 13.4, 8.7, 5.0 Hz, 2H), 1.70 – 1.52 (m, 14H), 1.43 (d, J
13
= 21.0 Hz, 18H), 1.01 (d, J = 6.9 Hz, 6H); C NMR (101 MHz, CDCl3): δ 170.9, 170.7, 154.7,
154.0, 136.3, 136.2, 116.4, 79.7, 79.6, 65.6, 65.1, 56.7, 46.6, 46.4, 43.5, 43.3, 39.3, 38.2, 37.2,
36.2, 35.8, 28.7, 28.6, 26.7, 25.7, 24.7, 19.9; Rf = 0.30 (4:6 EtOAc:hexanes); [α]D20 = – 6.5º (c =
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0.3, CHCl3); HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C19H32N2O3H+ 337.2491; Found
337.2491.
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2-((3R,5S)-3-methyl-5-(piperidine-1-carbonyl)pyrrolidin-2-yl)acetic acid (S-05)
To a flame-dried 10 mL round bottom flask under Ar (w/ stir bar) was added NaH (22 mg, 0.55
mmol, 2.0 eq) followed by 3.0 mL anhydrous THF (0.2M). tert-Butyl diethylphosphonoacetate
(130 µL, 0.55 mmol, 2.0 eq) was added dropwise, and the resulting solution was stirred for 1 h.
Amide 23 (86 mg, 0.27 mmol, 1.0 eq) was added to a flame-dried scintillation vial and diluted
with 550 µL anhydrous THF (0.1 M final concentration). The resulting solution was added to the
round bottom flask dropwise. After stirring for 5 h, the reaction was quenched with sat. aq.
NH4Cl (2 mL). The resulting mixture was diluted with 10 mL EtOAc. The aq. layer was
extracted with EtOAc (3 x 5 mL). The organics were then washed with sat. aq. NaCl, dried over
MgSO4, filtered, and concentrated in vacuo. The crude oil was then dissolved in 800 µL DCM
(0.3M). The solution was cooled to 0 °C using an ice bath, and treated with 270 µL TFA (1.0 M
final TFA concentration). The reaction was heated at 80 °C using an oil bath for 12 h. The
reaction was concentrated in vacuo and purified using Dowex 50WX8 resin (5 grams). The resin
was charged with 1 N HCl (until pH = 1) and washed with H2O (until pH = 6–7). The crude
material was added to the resin in H2O, washing with MeCN then H2O. 1 N NH4OH was then
used to wash the resin until S-05 eluted completely as judged by TLC (Note: TLC plates were
run in 2:8 DCM:MeOH and dried for 1 min to remove NH4OH prior to staining with ninhydrin).
The fractions were concentrated in vacuo affording S-05 (44 mg, d.r. = 1.7:1.0, 63% over two
1
steps) as a white solid. H NMR (400 MHz, CD3OD): δ 4.75 (dd, J = 9.7, 6.4 Hz, 2H), 4.70 –
4.61 (m, 1H), 3.96 (dt, J = 9.6, 5.7 Hz, 1.6H), 3.67 – 3.51 (m, 6H), 3.45 (t, J = 5.7 Hz, 5H), 2.71
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(dd, J = 16.8, 3.9 Hz, 1H), 2.62 – 2.54 (m, 1H), 2.53 – 2.36 (m, 5H), 2.27 (ddd, J = 13.1, 9.7, 7.3
Hz, 1.7H), 2.19 – 2.00 (m, 5H), 1.75 – 1.52 (m, 16H), 1.12 (d, J = 5.1 Hz, 3H), 1.06 (d, J = 7.0
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Hz, 5H); C NMR (101 MHz, CD3OD): δ 177.6, 176.9, 168.3, 167.9, 64.7, 62.0, 57.8, 57.1,
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47.4, 47.3, 44.8, 44.7, 37.5, 37.4, 37.0, 36.9, 36.0, 33.2, 27.2, 26.5, 25.3, 16.2, 13.3; [α]D = –
53.1º (c = 0.3, MeOH); mp = 59 °C; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H22N2O3H+
255.1709; Found 255.1719.
(2S,4R,5S)-4-methyl-2-(piperidine-1-carbonyl)-1-azabicyclo[3.2.0]heptan-7-one (27)
To a flame-dried 10 mL round bottom flask (w/ stir bar) under Ar was added 2-chloro-1-methyl-
pyridinium iodide (Mukaiyama reagent) (0.119 g, 0.47 mmol, 4.0 eq), 1.5 mL anhydrous MeCN,
and TEA (130 µL, 0.93 mmol, 8 eq). The mixture was heated to 70 °C in an oil bath with stirring
for 30 min. To a second flame-dried 10 mL round bottom flask (w/ stir bar) under Ar was added
S-05 (29.6 mg, 0.12 mmol, 1.0 eq) and 1.5 mL anhydrous MeCN. The resulting solution was
subsequently heated to 70 °C in an oil bath with stirring for 30 min. The solution from the first
round bottom flask was taken up in a syringe and added to the second round bottom flask
dropwise. The first round bottom flask was rinsed with 3 mL anhydrous DCM, and the resulting
solution was added dropwise to the second round bottom flask. After 15 min, the reaction was
gradually cooled to 23 °C by turning off the heat but leaving the reaction in the oil bath. After
stirring for 16 h, the reaction was concentrated in vacuo. The resulting oil was dilute with 10 mL
EtOAc and 10 mL sat. aq. NaCl. The aq. layer was extracted with EtOAc (3 x 5 mL). The
organics were then washed with sat. aq. NaCl, dried over MgSO4, filtered, and concentrated in
vacuo. The resulting crude material was purified by silica gel chromatography using gradient
elution (6:4 EtOAc:hexanes to 7:3 EtOAc:hexanes), affording 27 (11 mg, d.r. = 5.0:1.0, 41%) as
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a white film. 1H NMR (400 MHz, CDCl3): δ 4.66 (dd, J = 7.4, 5.0 Hz, 5H), 4.58 (dd, J = 8.8, 7.5
Hz, 1H), 3.92 (ddd, J = 6.7, 5.3, 2.6 Hz, 4H), 3.62 (ddd, J = 17.2, 8.7, 5.0 Hz, 8H), 3.57 – 3.45
(m, 17H), 3.43 – 3.35 (m, 3H), 3.23 – 3.16 (m, 1H), 3.08 – 2.99 (m, 5H), 2.76 (dd, J = 16.0, 2.6
Hz, 5H), 2.70 – 2.54 (m, 12H), 1.94 – 1.78 (m, 16H), 1.77 – 1.68 (m, 11H), 1.68 – 1.43 (m,
58H), 1.21 (d, J = 6.2 Hz, 2H), 1.15 (d, J = 6.5 Hz, 2H), 1.10 (d, J = 6.7 Hz, 2H), 1.01 (d, J = 7.1
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Hz, 14H); C NMR (101 MHz, CDCl3): (Note: Only the major epimer is clearly observable) δ
177.3, 168.1, 56.7, 55.6, 46.7, 43.7, 40.8, 37.4, 36.8, 33.2, 26.5, 25.7, 24.8, 24.6, 23.5, 15.5; Rf =
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0.30 (7:3 EtOAc:hexanes); [α]D = – 24.6º (c = 0.5, CHCl3); HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C13H20N2O2H+ 237.1603; Found 237.1577.
(2S,4R)-1-(tert-butoxycarbonyl)-4-methylpyrrolidine-2-carboxylic acid (29)
To a flame-dried 50 mL round bottom flask under Ar (w/ stir bar) was added N,O-
dimethylhydroxylamine hydrochloride (0.64 g, 6.56 mmol, 3.0 eq) and 6.6 mL anhydrous THF.
The suspension was cooled to 0 °C using an ice bath and AlMe3 (2 M in PhMe, 3.3 mL, 6.56
mmol, 3.0 eq) was added via syringe pump over 30 min. The resulting solution was stirred for 1h
at 23 °C, then cooled to –30 °C using a dry ice/acetone bath. Lactone 7 (0.501 g, 2.19 mmol, 1.0
eq) was added to a dry scintillation vial, diluted with 4.3 mL anhydrous THF (0.5M), and added
dropwise over 10 min to the 50 mL round bottom at –30 °C. After stirring 30 min, the reaction
was then quenched at –30 °C with 20 mL 10% (w/w) aq. Rochelle’s salt (20mL) and Et2O
(20mL). The resulting mixture was allowed to stir at 23 °C for 30 min, diluted with H2O and
filtered over celite. The aq. layer was then extracted with Et2O (3 x 20 mL). The organics were
washed with sat. aq. NaCl (50 mL), dried over MgSO4, filtered, and concentrated by air stream.
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The resulting oil was placed under Ar, dissolved in 22 mL anhydrous THF (0.1 M), and cooled
to –78 °C using a dry ice/acetone bath. After treating with TEA (610 µL, 4.37 mmol, 2.0 eq) and
MsCl (186 µL, 2.40 mmol, 1.1 eq), dropwise, the reaction was stirred for 1 h at – 78 °C then
allowed to warm to 23 °C. KOtBu (1.23 g, 10.93 mmol, 5.0 eq) was then added, and the reaction
was allowed to stir 12 h. The reaction was filtered over celite and concentrated in vacuo. The
resulting material was diluted with 11 mL H2O (0.2 M final concentration) and treated with
LiOH (0.523 g, 21.85 mmol, 10.0 eq). The resulting slurry was heated to 80 °C for 19 h. The
material was then filtered over celite. The aq. layer was extracted with Et2O and acidified to pH
= 1 using aq. HCl (1N). The solution was then extracted with DCM (3 x 20 mL). The organics
were washed with sat. aq. NaCl, dried over MgSO4, filtered, and concentrated in vacuo. The
resulting crude material was purified by silica gel chromatography using gradient elution (DCM
to 5:95 MeOH:DCM), affording 29 (0.222 g, 44% over three steps, mixture of rotamers) as a
slightly yellow oil. 1H NMR (400 MHz, CD3OD): δ 4.31 – 4.20 (m, 2H), 3.64 (m, 2H), 3.31 (p, J
= 1.7 Hz, 12H), 2.92 (m, 2H), 2.41 – 2.32 (m, 2H), 2.12 – 2.07 (m, 2H), 1.95 – 1.85 (m, 2H),
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1.46 (s, 9H), 1.42 (s, 9H), 1.06 – 1.03 (m, 6H); C NMR (101 MHz, CD3OD): δ 155.9, 81.4,
81.2, 60.7, 54.2, 39.4, 38.7, 33.2, 32.5, 31.1, 28.7, 28.6, 17.6, 17.5; Rf = 0.30 (5:95
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MeOH:DCM); [α]D = – 60.8º (c = 0.4, MeOH); HRMS (ESI-TOF): [M+H]+ - [CO2 +
CH2C(CH3)2] Calcd for C6H11NO2H+ 130.0868; Found 130.0861.
(2S,4R)-4-methylpyrrolidinium-2-carboxylic acid 2,2,2-trifluoroacetate (S-06)
To a 1-dram vial (w/ stir bar) was added 30 (1.7 mg, 0.0074 mmol, 1.0 eq), 100 µL DCM, and
7.4 µL TFA. The reaction was stirred at 23 °C for 48 h. The reaction was then concentrated in
1
vacuo. The resulting mass and yield were not obtained. H NMR analysis of this crude material
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shows only one compound. 1H NMR (400 MHz, CD3OD): δ 4.31 (dd, J = 9.5, 4.3 Hz, 1H), 3.54
(dd, J = 11.3, 7.3 Hz, 1H), 2.83 (dd, J = 11.4, 8.8 Hz, 1H), 2.39 (ddt, J = 19.8, 11.9, 6.9 Hz, 2H),
2.06 – 1.91 (m, 1H), 1.13 (d, J = 6.4 Hz, 3H).
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Supplementary figures and spectral data for all compounds
Crystallographic data for 19
Acknowledgments
Financial support for this work was generously provided by The Scripps Research Institute. We
thank C. A. Reiher and R. A. Shenvi for helpful discussions on tertiary lactone opening. We are
grateful to A. L. Rheingold, C. E. Moore, and M. Gembicky (University of California, San
Diego) for x-ray crystallographic analysis. We thank the Shen lab, the Disney lab, the Kodadek
lab, and the Roush lab for generous access to their instrumentations.
References
1. Rane, R.; Sahu, N.; Shah, C.; Karpoormath, R. Curr. Top. Med. Chem. 2014, 14, 253–
273.
2. Kobayashi, J.; Kanda, F.; Ishibashi, M.; Shigemori, H. J. Org. Chem. 1991, 56,
4574−4576.
3. Hager, A.; Vrielink, N.; Hager, D.; Lefranc, J.; Trauner, D. Nat. Prod. Rep. 2016, 33,
491–522.
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