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Letter
Proximity-Induced Reactivity and Product Selectivity with a Rationally Designed Bifunctional Peptide Catalyst Michael J. Kinghorn, Gabriel A. Valdivia Berroeta, Donalee R. Chantry, Mason S. Smith, Chloe C. Ence, Steven R. E. Draper, Jared S. Duval, Bryan M. Masino, Samuel B Cahoon, Rachael R. Flansburg, Cory J. Conder, Joshua L. Price, and David J. Michaelis ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b02699 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017
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Proximity-Induced Reactivity and Product Selectivity with a Rationally Designed Bifunctional Peptide Catalyst Michael J. Kinghorn, Gabriel A. Valdivia Berroeta, Donalee R. Chantry, Mason S. Smith, Chloe C. Ence, Steven R. E. Draper, Jared S. Duval, Bryan M. Masino, Samuel B. Cahoon, Rachael R. Flansburg, Cory J. Conder, Joshua L. Price,* and David J. Michaelis*
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 *Corresponding author email:
[email protected];
[email protected] Abstract Cooperative catalytic systems are making significant advances in modern organic synthesis due to the potential to combine multiple catalytic cycles or enable enzyme-like proximity effects. We report the rational design of a bifunctional helical peptide catalyst that displays an imidazolidinone catalyst in close proximity to a thiourea binding site and enables proximityenhanced reactivity and selectivity. The helical structure of the peptide and the binding of both reactants are shown to be essential for enhanced reactivity in Diels-Alder and indole alkylation reactions, and up to 28,000 catalyst turnovers are achieved. A variety of Lewis basic functional groups facilitate binding and proximity-enhanced reactivity and product selectivity is observed that cannot be achieved in the absence of the peptide template.
The rational design of small organic catalysts that engage multiple substrates in close
proximity and enable enzyme-inspired reactivity represents a significant pursuit in modern organic synthesis.1–13 Such catalysts can achieve reactivity and selectivity that surpasses that of monofunctional catalysts by capitalizing on proximity-accelerated reactivity, substrate-
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preorganization, and recognition-driven selectivity, which traits are often observed in enzyme catalysis. This approach to catalyst design complements work in rational enzyme design14–17 and provides an alternative method for developing enzyme-like catalysts that take advantage of reactions and mechanisms that do not occur in biology.18 In addition, organic catalysts commonly display high substrate tolerance across a broad range of chemical structures, which is often a significant limitation with top-down approaches to reengineering natural enzymes.19,20 Small polypeptides that fold into well-defined secondary and tertiary structures represent an attractive platform for developing synthetic enzyme-inspired catalysts. Representative examples of this strategy include coiled-coil assemblies of α-helical peptides that juxtapose catalytic residues,21–24 stereoselective polyamide catalysts that rely on multiple hydrogenbonding interactions,25–28 and helical or b-turn peptides that scaffold two or more catalytic groups.29–39 One significant limitation to these systems to date is their reliance on natural amino acid residues such as cysteine, histidine, and lysine to enable catalysis. The Ball group recently reported a helical peptide containing a non-natural dirhodium carboxylate that enables siteselective protein functionalization via a metal carbene transfer mechanism.40 Maayan has also used a peptoid backbone to scaffold phenanthroline-copper and TEMPO catalysts required for aerobic oxidations.4 What is much rarer is the use of a small structurally-defined polypeptide to scaffold multiple non-natural organic catalysts (e.g. Lewis acids, transition metals, organocatalysts) and facilitate both proximity-induced reactivity and recognition-driven selectivity.41–44 Here we show that placement of a catalytic site (imidazolidinone iminium catalyst) in close proximity to a binding site (thiourea hydrogen bonding group) one turn apart
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on a helical peptide leads to accelerated reactivity and unique product selectivity in the context of known catalytic Diels-Alder cycloaddition and indole alkylation reactions (Figure 1).45,46
Figure 1. Rationally designed bifunctional helical peptide catalyst.
We hypothesized that placing a thiourea near a chiral imidazolidinone along a rigid scaffold might allow the thiourea to recruit a carbamate-functionalized diene (Figure 1, blue) to the immediate vicinity of an imidazolidinone-activated α,β-unsaturated aldehyde (Figure 1, red). The resulting Diels-Alder reaction would then benefit from proximity-induced rate acceleration.1, 47 α-Helical peptides represent an ideal scaffold for testing this hypothesis because their regular pattern of i to i+4 H-bonding brings side chains at these positions within ~7 Å of each other, which we envisioned would be close enough to observe proximity effects. Importantly, such proximity effects have been observed with other thiourea-amine bifunctional catalysts.13,48–51 For our helical scaffold, we chose an organic-soluble 11-residue peptide p1, in which 2-aminoisobutyric acid (Aib, B) residues promote helical secondary structure (see below).52,53 The desired imidazolidinone (X) and thiourea (Z) groups at the 2 and 6 positions respectively were incorporated via on-resin side-chain modification following solid-phase synthesis (p2). Residue X was prepared by modifying the Lys2 ε-amino group with an arylisocyanate. Likewise, residue Z
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was prepared by from azidolysine via the copper-catalyzed azide-alkyne cycloaddition with an alkyne-linked imidazolidinone. For the purpose of control studies, we synthesized peptides containing only Z or X (p3 and p4), a bifunctional random coil peptide p5 containing helixbreaking prolines54 in the place of Aib residues, and non-peptide bound catalysts 1 and 2.
p1 p2 p3 p4 p5
AcNH– V AcNH– ⋅ AcNH– ⋅ AcNH– ⋅ AcNH– ⋅
A X ⋅ X X
L ⋅ ⋅ ⋅ ⋅
B ⋅ ⋅ ⋅ P
A Z Z ⋅ Z
V ⋅ ⋅ ⋅ ⋅
L ⋅ ⋅ ⋅ ⋅
B ⋅ ⋅ ⋅ P
V ⋅ ⋅ ⋅ ⋅
A ⋅ ⋅ ⋅ ⋅
L –NHMe ⋅ –NHMe ⋅ –NHMe ⋅ –NHMe ⋅ –NHMe CF3
H N
O
N
NH
S
HN
O
1
X
O CF3
O
N H
N H
HN
NH
S N H
2
CF3
N N H
CF3
HN
O
N O
Z
N
N
Figure 2. Peptide sequences and catalyst structures. Amino acids are abbreviated according to the standard oneletter code except for X and Z, whose structures are as indicated, and B, which represents 2-aminoisobutyric acid (Aib).
The circular dichroism (CD) spectra of p2, p3, and p4 closely resemble that of p1 (see Figure S2), suggesting that our derivatives adopt α-helical conformations. Non-sequential NOEs observed in 2D NMR studies of p2 are similarly consistent with α-helical secondary structure. Importantly, NOEs between side chains X and Z on p2 confirm that the thiourea and imidazolidinone groups are close in space on the helix (see Figure S3–S6). In contrast, the CD spectrum of proline-containing p5 is not consistent with helical structure, suggesting that p5 adopts a random-coil conformation as desired. We first explored the relative abilities of bifunctional peptide p2, monofunctional peptide p3, and catalyst 1 to catalyze the known Diels-Alder reaction45,55 between carbamate-containing
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diene 3 and α,β-unsaturated crotonaldehyde (Figure 3). The p2-catalyzed reaction at 100 mM diene concentration reaches 88% conversion after 48 hours, which is substantially higher than observed for the uncatalyzed reaction (3.4% conversion). In contrast, the control reactions with imidazolidinone-containing peptide p3 or with p3 and thiourea containing peptide p4 reached only 7.1% and 7.6 % conversion, respectively, in the same time. Non-peptide bound imidazolidinone 1 also provide lower conversion than p2, individually (46% conversion) or in combination with thiourea 2 (35% conversion). The disparity in reactivity between p2 and either p3 or 1 is further enhanced as the concentration of diene 3 is lowered to 10 mM and 1 mM. Also of particular note is the lower reactivity of the imidazolidinone peptide p3 with respect to catalyst 1 (7% vs 46% conversion at 100 mmol 3, respectively). This lower reactivity indicates that incorporation of the imidazolidinone catalyst onto the helical peptide scaffold is actually detrimental to reactivity, and that the presence of thiourea residue X is essential to rescuing reactivity. In addition, our initial conversion studies appear to show first order dependence on catalyst concentration, as expected for bifunctional catalysts, and more detailed kinetic studies are currently underway. These data are consistent with our hypothesis that binding both substrates on the helix is essential for proximity-accelerated reactivity. O OHC +
HN
NHCBZ OBn
20 mol% catalyst
3
NO2Me:H2O 95:5 –15 ºC, 48 h
Me
3 equiv
OHC Me
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Figure 3. Reactivity of bifunctional peptide catalyst p2 vs monofunctional counterparts as a function of diene concentration. % Conversion determined by mass spectrometry of crude reactions. Cbz = carboxybenzyl. Absolute and relative configuration of 4 assigned by comparison with published re-sults,14 and all other assignments by analogy.
We next tested the reactivity of non-helical peptide p5 containing both X and Z residues, where helicity is interrupted by introduction of proline in place of helix-inducing Aib residues. We found that the reactivity of p5 at 10 mM diene 3 dropped to nearly zero (2% conv, 48 h), whereas p2 provided 80% conversion in the same time. In fact, the reactivity of p5 mimicked that of monofunctional catalysts p3 and 1, which produced 4% and 6% product respectively in the same time. We also tethered catalysts 1 and 2 via a linear PEG-4 (polyethylene glycol) linker, mimicking the distance between the two catalysts on the peptide. In this case as well, reactivity similar to catalyst 1 alone was observed and no proximity-induced rate enhancement occurred (see supporting information for details). These results suggest that the well-defined helical structure of p2 is directly responsible for enabling proximity effects in the Diels-Alder reaction and that simply tethering two catalytic groups four-residues apart is not sufficient to achieve the same reactivity. Our final control study confirmed the importance of binding the diene partner to the thiourea residue X on p2. When cyclopentadiene 5 was employed in the Diels-Alder reaction, catalyst p2
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reached 32% conversion to 6 in 48 h, whereas imidazolidinone 1 achieved 70% conversion (Figure 4). Cyclopentadiene 5 lacks the binding site necessary to H-bond with thiourea X and thus cannot participate in cooperative binding. In fact, reactions with diene 5 catalyzed by p2 proceeded at essentially the same rate as those catalyzed by monofunctional peptide p3 (32% vs 34% conversion). nonbinding diene
OHC
20 mol% catalyst
+
NO2Me/H2O –15 ∞C, 48 h
Me
5
catalyst % conv.
OHC Me
1 p2 p3
6
70% 32% 34% 1
Figure 4. Reactivity of catalysts 1, p2, and p3 with nonbinding diene 5. Conversion recorded by H NMR analysis.
The ability of our bifunctional catalyst to discriminate between dienes that can or cannot bind thiourea residue X led us to question whether catalyst p2 could overcome the inherent selectivity of the imidazolidinone catalyst for electron-rich dienes and instead enable binding-induced selectivity. Indeed, in the competition between diene 3 and cyclopentadiene 5 with a limiting amount of crotonaldehyde, catalyst p2 preferentially recruited diene 3 to provide product 4 in 16:1 selectivity (Figure 5). In stark contrast, imidazolidinone catalyst 1 reacted with slight preference for cyclopentadiene, giving the opposite product (6) in 1.4:1 selectivity. NHCBZ NHCbz
OHC
3 p2 or 1
OHC
+ Me
5
NO2Me/H2O 4 ∞C, 48 h
1 mmol 2 mmol each
4
Isolated Yields 1 mol% 10 mol% p2 1 48
19
Me OHC
6 Me
3
27
(16:1)
(1:1.4)
Figure 5. Competition between dienes 3 and 5 for Diels-Alder reactivity with catalyst p2. Yields determined by 1
isolation (4) and by H NMR analysis of 6 by comparison with an internal standard due to the volatility of 6.
In order to test the efficiency of our catalyst, we conducted the Diels-Alder reaction at 1 M diene concentration with 0.001 mol% catalyst and observed 28% conversion to product in 48 h,
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corresponding to up to 28,000 turnovers. No product (>5% conversion) was detected in the absence of catalyst under the same conditions. We also performed the reaction to 100% conversion with 0.1 mol% p2 and obtained a 63% isolated yield of the product in 89% ee. The high ee of the product confirms that the chiral imidazolidinone is indeed catalyzing the transformation. When the same reaction was conducted with 10 mol% of imidazolidinone catalyst 1 (100 times catalyst loading), a similar 57% yield was obtained with 77% ee, suggesting that the peptide scaffold in p2 significantly improves the inherent enantioselectivity of the imidazolidinone catalyst. We next investigated the substrate scope of catalyst p2 and confirmed that a variety of Lewis basic functional groups can be recruited by the thiourea side chain. For example, Diels-Alder products containing carbamates (4), carbonates (7), ureas (8), and simple esters (9) are all generated in high yield using p2 at 1 mol% catalyst loading (Figure 6). In contrast, very little product was generated under the same conditions with catalyst 1. O HN
O OBn
OHC
O
OHC
O OBn
HN
O NHBn
OHC
O
Me
OHC
Me
4 p2 = 63% 1 =
