Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme

Article abstract of DOI:10.1021/jacs.9b01596

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,β-unsaturated-δ-lactams via a tandem C-H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp?) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11-33 times more reactivity.

Full text of DOI:10.1021/jacs.9b01596

Communication
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Asymmetric δ‑Lactam Synthesis with a Monomeric Streptavidin
Artificial Metalloenzyme
†
,§
‡,§
†
†,‡,§
†,‡
Isra S. Hassan, Angeline N. Ta, Michael W. Danneman,
Natthawat Semakul,
Matthew Burns,† Corey H. Basch, Vanessa N. Dippon, Brian R. McNaughton,*
and Tomislav Rovis*
,‡
†
,‡
,
†,‡
†
Department of Chemistry, Columbia University, New York, New York 10027, United States
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States
‡
*
S Supporting Information
direct union of acrylamide hydroxamate esters with alkenes has
15
ABSTRACT: Reliable design of artificial metalloenzymes
ArMs) to access transformations not observed in nature
not been disclosed.
16
(
In collaboration with the Ward group, we have previously
described a tetrameric streptavidin (tSav) system containing a
biotinylated Rh(III) cofactor for the asymmetric synthesis of
dihydroisoquinolones using benzhydroxamate esters and
remains a long-standing and important challenge. We
report that a monomeric streptavidin (mSav) Rh(III)
ArM permits asymmetric synthesis of α,β-unsaturated-δ-
lactams via a tandem C−H activation and [4+2]
annulation reaction. These products are readily derivat-
ized to enantioenriched piperidines, the most common N-
heterocycle found in FDA approved pharmaceuticals.
Desired δ-lactams are achieved in yields as high as 99%
and enantiomeric excess of 97% under aqueous conditions
at room temperature. Embedding a Rh cyclopentadienyl
17
acrylate partners (Figure 1ii). In concurrent work, Cramer
reported a small-molecule chiral Rh(III) catalyst for a
complementary coupling reaction involving styrene partners
18
(
Figure 1iii).
Table 1. Reaction Optimization and Controls
(
Cp*) catalyst in the active site of mSav results in
improved stereocontrol and a 7-fold enhancement in
reactivity relative to the isolated biotinylated Rh(III)
cofactor. In addition, mSav-Rh outperforms its well-
established tetrameric forms, displaying 11−33 times
more reactivity.
NMR yield
enantiomeric
b
a
entry
catalyst
(%)
excess (%)
1
2
3
Cp*RhCl2
25
15
9
0
0
−26
0
92
91
82
Cp*biotinRhCl2
wt-tSav:Cp*biotinRhCl2
N118K-K121E-tSav:Cp*biotinRhCl
wt-mSav:Cp*
wt-mSav:Cp*
wt-mSav:Cp*biotinRhCl
iperidines are the most common N-heterocycle found in
FDA approved drugs. Strategies to assemble this
c
4
5
6
7
3
2
1
P
d
biotin
RhCl2
RhCl2
44
99
58
biotin
important structural motif are common but often involve
cyclization, which necessitates the preassembly of an acyclic
2
2
precursor. Two-component approaches such as cycloadditions
2a (3.0 μmol), 3a (1.5 μmol), catalyst, in 200 μL of acetate buffer
62.5 mM NaOAc, 100 mM NaCl, pH 7.4) with 3 μL of MeOH.
Conversion and yield determined by H NMR analysis relative to a
trimethyl(phenyl)silane internal standard. Enantiomeric excess
determined by HPLC analysis. 1 mol % catalyst. 200 μL of NaCl
buffer (100 mM NaCl, pH 7.4) used.
(
or annulations are inherently more attractive. A handful of
recent [4+2] cycloaddition approaches have been devised,
a
1
b
3
−5
6
using N-heterocyclic carbene,
cinchona alkaloid, chiral
c
d
7
8,9
phosphoric acid or metal catalysis. Although each strategy is
useful, they are limited to specific classes of precursors and
substitution patterns. An alternative approach is to develop a
methodology that enables access to α,β-unsaturated-δ-lactams
The most common ArM platform developed to date is the
biotin-tetrameric (strept)avidin (biotin-tSav) system, pio-
neered by Whitesides and Ward (Figure 1a). These
10,11
from simple precursors under mild conditions.
In this
19
20
context, the coupling of acrylamides with abundant α-olefins
via C−H activation and annulation would provide direct access
to α,β-unsaturated-δ-lactams in a single step.
−14
ArMs utilize high affinity (up to K ∼ 10 M) interactions
D
between tSav and biotin−metal conjugates. tSav-based ArMs
have been utilized in an increasing number of transition-metal
1
4
Rh(III) catalysis has been extensively demonstrated to
mediated transformations. Herein, we introduce a new ArM
platform based on monomeric streptavidin (mSav) and
demonstrate its use in the direct enantioselective coupling of
2
mediate C−H activation reactions involving C(sp )−H bonds
12
proximal to a directing group. The subsequent functionaliza-
tion with alkenes is less well-developed but has been
13
documented for benzhydroxamate esters and unsaturated
Received: February 11, 2019
1
4
oxime esters delivering N-heterocycles in good yield. The
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b01596
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 1. (a) i. tetrameric streptavidin (tSav) ArM; ii. Previously developed tSav Rh(III) benzhydroxamate coupling; iii. Cramer’s enantioselective
Chiral Cp variant. (b) iv. monomeric streptavidin (mSav) ArM. v. Target transformation (this work). [tSav (PDB ID: 3RY1) and mSav (PDB ID:
4
JNJ) were complexed with the biotin cofactor in AutoDock and rendered in PyMOL.]
Scheme 1. (a) Methyl Acrylamide Styrene Scope; (b) Extended Acrylamide Styrene Scope
a
Reaction conditions: 2a (3.0 μmol), 3 (1.5 μmol), catalyst, in 200 μL of acetate buffer (62.5 mM NaOAc, 100 mM NaCl, pH 7.4) with 3 μL
b
1
MeOH, at 25 °C for 72 h. Yields determined by H NMR analysis relative to a trimethyl(phenyl)silane internal standard. Enantiomeric excess
determined by HPLC analysis. PMP = para-methoxyphenyl.
acrylamide hydroxamate esters and styrenes (Figure 1v). The
reaction allows rapid access to piperidines, the most common
N-heterocycle found in FDA-approved pharmaceuticals.
Initial investigations examined the coupling of methacryla-
22
mide 2a with styrene 3a. The use of Cp*Rh(III) as a catalyst
under aqueous buffer conditions delivers 4aa in modest yield
1
,21
B
DOI: 10.1021/jacs.9b01596
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
23
(
entry 1, Table 1). The use of biotinylated Rh cofactor as
Scheme 2. (a) Proposed Mechanism; (b) Labelling
catalyst provides 4aa in 15% yield. Incorporation of the
cofactor into wt tSav leads to further diminished yields and
very low selectivities (entry 3, Table 1). Similarly, a mutant
tSav (N118 K/K121E) that proved to be a highly reactive ArM
Experiment; (c) Mutational Study
14
in our previous work did not provide the desired δ-lactam
4aa) in appreciable yield (Table 1, entry 4). In an effort to
(
improve the reaction, we turned our attention to monomeric
streptavidin (mSav, Figure 1-iv). We reasoned that the use of
mSav, with a more exposed Rh binding site relative to tSav,
could serve as a competent ArM template, and may simplify
ArM tuning and analysis. Like tSav, biotin is bound tightly by
2
4−26
mSav (K ∼ 2 nM
) but has been engineered to resist
D
tetramerization by replacing hydrophobic amino-acid residues
at the barrel−barrel interface with charged ones.
biotin
In the event, 1 mol % wt-mSav:Cp*
RhCl ArM in
2
acetate buffer enables the coupling of acrylamide (2a) and
para-methoxystyrene (3a) to provide the desired δ-lactam
(
1
4aa) in 44% yield and 92% enantiomeric excess (ee), (Table
, entry 5). A modest increase in metalloenzyme catalyst
27
loading results in substantially higher yield and virtually
identical selectivity, delivering the desired δ-lactam (4aa) in
9
9% yield and 91% ee (Table 1, entry 6). The use of 100 mM
NaCl in place of acetate buffer leads to 58% yield (Table 1,
entry 7).
The wt-mSav:Cp*biotinRhCl2 catalyzed reaction proved
broadly tolerant to the coupling partners employed (Scheme
1
). With respect to the styrene partner (Scheme 1a),
enantioselectivities are best with para-substituted styrenes,
regardless of electronic character. Meta-substituted styrenes are
also tolerated, affording good to high enantioselectivities, while
a single ortho-substituted styrene leads to somewhat decreased
selectivity. Styrene itself is a poor substrate, proceeding in
modest selectivity and yield (4ab). Importantly, all substrates
give the desired δ-lactam products as single regioisomers.
Substitution on the acrylamide is well tolerated regardless of
steric demand affording product with enantioselectivities that
match the corresponding methacrylamide system (Scheme
a
Reaction conditions: 2a (3.0 μmol), 3 (1.5 μmol), catalyst, in 200
μL of acetate buffer (62.5 mM NaOAc, 100 mM NaCl, pH 7.4) with
b
1
3
μL of MeOH, at 25 °C for 72 h. Yields determined by H NMR
analysis relative to a trimethyl(phenyl)silane internal standard.
ArM featuring wt-mSav, the Y112A mutant provides the
desired product in low yield and enantiomeric excess (37% and
61%, respectively). This observation is consistent with its likely
role as a rigidifying element through π-stacking to the Cp
framework on the catalyst.
reactivity and selectivity, Y112A maintains affinity for biotin
(see Supporting Information).
In order to derivatize the resulting δ-lactam products into
piperidines, the coupling of 2a and 3a to provide 4aa was
performed at a 0.15 mmol scale providing identical results to
the reaction performed on a 1.5 μmol scale (99% yield, 91%
ee) (Scheme 3a). Hydrogenation of 4aa affords the reduced
lactam 6aa in 99% yield and 10:1 dr. Subsequent reduction of
6aa with LiAlH furnishes the desired piperidine 5aa in 81%
yield and 7:1 dr (Scheme 3).
Indeed, the derivatization can proceed under exceedingly
mild reduction conditions. Treatment of a range of δ-lactams
(6) formed in good diastereoselectivity following hydro-
genation with BH ·SMe provides the corresponding piper-
1
b). However, aryl- and alkoxy- substitution results in
diminished yields (4ca, 4ea and 4ed).
28
17,29
A plausible catalytic cycle for this reaction is proposed in
Scheme 2. Metalation of the amide by rhodium generates
intermediate I. C−H activation occurs, presumably via a
concerted-metalation deprotonation (CMD) mechanism,
providing five-membered rhodacycle II. A deuterium labeling
experiment illustrates that the C−H activation step is
reversible, suggesting that the concerted metalation/deproto-
nation (CMD) is not the turnover limiting step (Scheme 2b).
Subsequent alkene coordination and migratory insertion would
give seven-membered rhodacycle IV. Available evidence
suggests this step is the enantiodetermining event. N−O
bond cleavage and reductive elimination then occurs to form
transient Rh(III) intermediate V. Protodemetalation regener-
Despite significant decreases in
4
ates the Rh(III) catalyst and closes the catalytic cycle.
biotin
While the Cp*
RhCl cofactor alone delivers the desired
2
3
2
δ-lactam (4aa) with no appreciable selectivity (0% ee, Table 1,
entry 2), its significantly reduced reactivity was a surprise (15%
yield with 3 mol % catalyst loading, compared to 99% yield
with an equivalent of the mSav artificial metalloenzyme). To
begin to evaluate the molecular dictates of reactivity and
idines in good yield and comparable diastereoselectivity to the
LiAlH reduction (Scheme 3b,c). Notably, these reduction
conditions are tolerant of ester functionalities (5fa) with a
slight erosion of diastereoselectivity, likely due to competitive
imine/enamine tautomerization of an intermediate.
4
stereocontrol, we determined that mutation of tyrosine 112
In conclusion, we have developed an artificial metal-
loenzyme that efficiently catalyzes an enantioselective tandem
C−H activation and [4+2] annulation reaction to afford δ-
biotin
(
Y112), which neighbors the putative Cp*
Rh pocket has a
signficant effect on the transformation. In comparison to the
C
DOI: 10.1021/jacs.9b01596
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 3. (a) Preparative Scale δ-Lactam Synthesis and Piperidine Derivatization; (b) Lactam Scope; (c) Piperidine Scope
a
b
1
Reaction conducted at a 0.15 mmol scale. Derivatization performed on racemic products. Diastereoselectivity determined by H NMR analysis
c
relative to a trimethyl(phenyl)silane internal standard. Isolated yields. Reaction conducted with 5 equiv BH ·SMe and 5 equiv n-PrNH .
3
2
2
substituted lactams. This metalloenzyme accepts a diverse
array of acrylamide and styrene coupling partners. The mSav
metalloenzyme platform demonstrates superior reactivity
relative to its tSav counterpart and the free cofactor alone.
Efforts to understand the crucial factors responsible for
improved reactivity are currently underway.
ment for a DPST graduate scholarship. I.S.H. acknowledges
the National Science Foundation for a predoctoral fellowship.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b01596.
■
*
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(
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