Chemoselective and Site-Selective Reductions Catalyzed by a Supramolecular Host and a Pyridine-Borane Cofactor

Article abstract of DOI:10.1021/jacs.0c12479

Supramolecular catalysts emulate the mechanism of enzymes to achieve large rate accelerations and precise selectivity under mild and aqueous conditions. While significant strides have been made in the supramolecular host-promoted synthesis of small molecules, applications of this reactivity to chemoselective and site-selective modification of complex biomolecules remain virtually unexplored. We report here a supramolecular system where coencapsulation of pyridine-borane with a variety of molecules including enones, ketones, aldehydes, oximes, hydrazones, and imines effects efficient reductions under basic aqueous conditions. Upon subjecting unprotected lysine to the host-mediated reductive amination conditions, we observed excellent ?-selectivity, indicating that differential guest binding within the same molecule is possible without sacrificing reactivity. Inspired by the post-translational modification of complex biomolecules by enzymatic systems, we then applied this supramolecular reaction to the site-selective labeling of a single lysine residue in an 11-amino acid peptide chain and human insulin.

Full text of DOI:10.1021/jacs.0c12479

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Article
Chemoselective and Site-Selective Reductions Catalyzed by a
Supramolecular Host and a Pyridine−Borane Cofactor
Mariko Morimoto, Wendy Cao, Robert G. Bergman,* Kenneth N. Raymond,* and F. Dean Toste*
Cite This: J. Am. Chem. Soc. 2021, 143, 2108−2114
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ABSTRACT: Supramolecular catalysts emulate the mechanism of
enzymes to achieve large rate accelerations and precise selectivity
under mild and aqueous conditions. While significant strides have
been made in the supramolecular host-promoted synthesis of small
molecules, applications of this reactivity to chemoselective and
site-selective modification of complex biomolecules remain
virtually unexplored. We report here a supramolecular system
where coencapsulation of pyridine−borane with a variety of
molecules including enones, ketones, aldehydes, oximes, hydra-
zones, and imines effects efficient reductions under basic aqueous conditions. Upon subjecting unprotected lysine to the host-
mediated reductive amination conditions, we observed excellent ε-selectivity, indicating that differential guest binding within the
same molecule is possible without sacrificing reactivity. Inspired by the post-translational modification of complex biomolecules by
enzymatic systems, we then applied this supramolecular reaction to the site-selective labeling of a single lysine residue in an 11-
amino acid peptide chain and human insulin.
transferases (KATs) and methyltransferases (KMTs) as well as
tyrosinases,19,20 which has been exploited in the recent years
for site-selective protein bioconjugation.21−25 We thus sought
to develop a supramolecular host system that would emulate
not only the mechanism of action of enzymes but also their
remarkable breadth of reactivity ranging from small molecules
to complex protein scaffolds. Drawing inspiration from the
pyridine-based cofactor of ketoreductase enzymes
(KREDs),26−28 we report here a supramolecular host-mediated
pyridine-borane reduction that proceeds under basic, aqueous
conditions and allows reactivity with a range of small molecules
including enones, ketones, aldehydes, oximes, hydrazones, and
imines (Figure 1c). We then demonstrate the ability of this
reaction to withstand unprecedented increases in substrate
complexity, as we investigate applications in the site-selective
reductive amination of lysine in peptides and proteins (Figure
1d).
INTRODUCTION
■
Supramolecular host−guest catalysis exerts precise molecular
control over a reaction through a network of noncovalent
interactions within the host cavity, shielded from competing, or
even overwhelming, interactions with solvent and other
reagents.1−11 For decades, chemists have sought to utilize
these hosts as synthetic enzyme mimics to harness the high
selectivity and reactivity of enzymatic processes under mild
and aqueous conditions. Terpene cyclization reactions are one
such example, where Tiefenbacher and co-workers utilized a
resorcinarene host to cyclize geranyl acetate, a process typically
catalyzed by terpene synthase enzymes including 1,8-cineole
synthase (Figure 1a).12−15 Site-selective and chemoselective
transformations of small molecules are another hallmark of
enzymatic catalysis, such as the selective reduction/oxidation
reactions of cortisol and corticosterone by 11β-hydroxysteroid
dehydrogenase (11β-HSD) (Figure 1b).16 More recently, site-
selective transformations promoted by supramolecular hosts
have also been demonstrated, such as the prenyl functionaliza-
tion of geranyllinalool by Fujita and co-workers as well as
selective Rh-catalyzed hydrogenation of a linolenic acid
derivative by Toste and co-workers.17,18 These examples
represent important advances in the practical applicability of
these biomimetic hosts in small molecule synthesis.
RESULTS AND DISCUSSION
■
The highly anionic Ga4L6 host-1 has been shown to catalyze a
number of Lewis and Bronsted acid-activated organic trans-
formations such as the Nazarov cyclization,29 and more
Received: November 30, 2020
Published: January 20, 2021
In contrast to small molecule synthesis, however, the
application of supramolecular host−guest chemistry to the
selective modification of larger and significantly more complex
biomolecules such as proteins remains extremely rare. Protein
post-translational modification, on the other hand, is an
integral function of many enzymes including lysine acetyl-
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Table 1. Host-1-Catalyzed Pyridine−Borane Reduction of
Aliphatic Ketones
a
NMR yields representing an average of two consecutive runs.
Reaction heated to 50 °C.
b
Figure 1. (a) Cyclization of GPP to 1,8-cineol catalyzed by 1,8-cineol
synthase and a resorcinarene cage. (b) Site-selective oxidation/
reduction by 11β-HSD and selective alkene functionalization by host-
1 and a Pd6L4 cage. (c) This work: host-1-mediated reduction with a
pyridine−borane cofactor, which mimics the reactivity of ketor-
eductase enzymes. (d) Site-selective post-translation modification of
biomolecules by various enzymes and a rare example of lysine-
selective biomolecule functionalization by a supramolecular host.
general trend, shorter alkyl ketones resulted in significantly
higher yields (Table 1, entries 1−3) in the presence of catalytic
loadings of host-1, with reactivity decreasing as the hydro-
phobic alkyl group grows longer (Table 1, entries 4 and 5).
While a similar trend is observed for ketones with pendant
alcohol groups (Table 1, entries 6 and 7), the decline in
reactivity is less dramatic, particularly upon gentle heating of
the reaction mixture. These observations imply that while
hydrophobicity is required for the substrate to be encapsulated
within the host, disproportionately strong binding of the
substrate (as in the case for 2d and 2e) may be preventing
coencapsulation with the pyridine−borane reductant, which is
necessary for the reaction to proceed.
recently an aza-Darzens reaction,30 through hydrophobic-
effect-driven binding and electrostatic stabilization of cationic
intermediates and transition states. In view of these previous
results, it was hypothesized that a hydride reduction reaction
such as that illustrated in Figure 1c could be mediated by host-
1 via protonation of the substrate upon encapsulation. We first
sought to identify a reducing agent that would not only be
compatible with the host but would also result in minimal
background reactivity in the absence of Lewis or Bronsted acid
activators. A screen of various organic reducing agents
identified pyridine-borane as a suitable candidate due to its
inertness under basic aqueous conditions and hydrophobic-
effect-driven binding to host-1 (see Figure S1 and Table S1 in
the Supporting Information).
While pyridine-borane has been used as a mild reducing
agent in reductive aminations with formaldehyde under near-
neutral conditions, acidic media such as acetic acid are
generally required for more challenging reductions including
those of ketones and enones.31−33 In the presence of catalytic
loadings of host-1 and 1 equiv of borane, reduction of aliphatic
and cyclic ketones 2a−i to the corresponding alcohol products
3a−i was observed, despite pD 8 water as the solvent (Table
1). Upon performing control experiments where the reaction
was run in the presence of stoichiometric NEt4+ (added as the
NEt4Cl salt), a strongly binding inhibitor of host-1, only traces
to low yields of alcohol product were observed in each case
with the exception of cyclohexanone (Table 1, entry 9). As a
Encouraged by these initial results, α, β-unsaturated ketones
were also investigated. In addition to potential acceleration in
the rates of reduction, enones could provide insight into the
inherent selectivity of the host-catalyzed reaction for the two
reducible sites at the 1,2- and 1,4-positions. Upon subjecting
mono- and disubstituted enones (4a−e) to host-1-mediated
reduction conditions, the corresponding allyl alcohols 5a−e
were consistently observed as the major products in moderate
+
yields. Control experiments with NEt4 -blocked host-1
demonstrated unexpectedly high selectivities for the corre-
sponding saturated ketone products 6a−c for β-monosub-
stituted enones (Table 2, entries 1−3), yielding only trace
amounts of the allyl alcohol in each case. Disubstitution at the
β-position led to a marked decrease in background reactivity
(Table 2, entries 4 and 5), whereas the host-catalyzed reaction
preferentially yielded the allyl alcohol products 5d,e. The high
1,2-selectivity observed in the presence of the host may be
attributed to several factors: one possibility is that
coencapsulation of the enone substrate with pyridine−borane
enforces a conformation where the borane is oriented closer to
the carbonyl group than the more distal 1,4-site.34 Alter-
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Table 2. Host-1-Catalyzed Pyridine−Borane Reduction of
α,β-Unsaturated Ketones
Table 3. Host-1-Catalyzed Pyridine−Borane Reduction of
Oximes and Hydrazones
a
b
Yields represent an average of two consecutive runs. Yield obtained
a
NMR yields representing an average of two consecutive runs.
from CDCl3 extract due to lower water solubility of the product.
b
Selectivities determined by extracting the reaction with CDCl3 and
c
comparing to known spectral data. Reaction run in 15% methanol
due to low solubility of starting material under aqueous conditions.
(Scheme 1a). Notably, alcohol byproducts are not observed,
presumably due to rapid oxime formation outcompeting
natively, protonation of the carbonyl group, which is stabilized
within the host cavity, may accelerate the 1,2-reduction
pathway over the 1,4-pathway that occurs via polarization of
the C−O bond. These results thus demonstrate that
confinement within the host cavity not only accelerates the
rate of the reaction but also overrides the innate selectivity of
the uncatalyzed pathway.
Scheme 1. (a) Host-1-Mediated One-Pot Synthesis of
Substituted Hydroxylamines; (b) Reduction of a Decanoic
Acid Oxime, Which Proceeds Despite Partial Encapsulation
of the Substrate
We next turned our attention to oximes, which are more
resistant toward reduction, even by stronger reductants such as
NaBH4 under pH neutral conditions. Addition of metal Lewis
acid additives such as CuSO4, TiCl4, and InCl3 or Bronsted
acids such as acetic acid are typically required, which often
leads to over-reduction of the hydroxylamine product to the
amine.35−37 Pyridine−borane has been shown to exclusively
yield the corresponding hydroxylamine but again required
strongly acidic conditions for the reaction to take place at an
observable rate.32 We thus decided to investigate whether host-
1 could be used to facilitate mild and selective reductions of
oximes to the corresponding hydroxylamines.
Upon subjecting aryl and aliphatic aldoximes and ketoximes
to host-1-mediated reduction conditions, moderate to high
yields of the hydroxylamine products were observed, with no
over-reduction to the amine. Aryl hydrazone 8b (Table 3,
entry 2) also underwent rapid reduction to the corresponding
benzyl hydrazine product, albeit at lower yields due to
heterogeneous reaction conditions. Under the mild reaction
conditions acid-sensitive acetal protecting groups are also well
tolerated, leading to high yields of the protected hydroxyl-
amine product 9d (Table 3, entry 4). Further experiments
demonstrated that substituted hydroxylamine formation can
also occur in a one-pot fashion, where the ketoxime is formed
in situ by its ketone and hydroxylamine components to form 9e
ketone reduction. Blocked-host control experiments did not
yield any observable conversion to the desired hydroxylamine
product for any of the oxime substrates.
O-Alkylation of oxime 8e with an allyl group (8g), however,
led to a marked decrease in yield, even under host-mediated
conditions (Table 3, entry 7). In a 1:1 competition experiment
with O-methyl oxime 8f, this disparity in reactivity is evident,
as a 15:1 selectivity for 8f over 8g is observed after 3 h. This
result was attributed to size exclusion, where the increased
steric bulk and hydrophobicity of O-allyl oxime 8g inhibit
coencapsulation within the host cavity with pyridine−borane.
We hypothesized that functionalization of the substrate with
an anionic carboxylate group could attenuate the hydro-
phobicity of the substrate and encourage partial encapsulation,
facilitated by unfavorable Coulombic interactions between the
carboxylate group and the host. Maintaining reactivity for
2110
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Figure 2. (a) 19FNMR data depicting selective generation of ε-functionalized 12 under 1-catalyzed conditions and a mixture of α-functionalized 11
and 12 for NaCNBH3 and blocked-host conditions. (b) Competition reaction between Gly and BocLysOH under 1-catalyzed and blocked-host
conditions.
partially encapsulated substrates would be imperative for site-
selective applications of this host-catalyzed reduction to more
complex substrates. To test this hypothesis, decanoic acid
oxime 8h was synthesized and subjected to 1-catalyzed
conditions (Scheme 1b). Despite being two carbons longer
than 8g, hydroxylamine product 9h was observed in moderate
under host-1 mediated conditions, whereas a mixture of both
modified lysine (14) and glycine (13) products was observed
upon blocking the host cavity with an equivalent of NEt4Cl.
The high selectivity of host-1 for the reductive alkylation of
the ε-terminus of lysine led us to extend this strategy to the
site-selective modification of lysine over a reactive N-terminus
in peptide chains. While selective reductive amination of the
N-terminus with benzaldehyde has been shown to proceed by
using NaCNBH3 under acidic conditions, selective alkylation
of lysine is still relatively limited.38,39 A notable strategy by Rai
and co-workers involves addition of a Cu-acetylide into an in
situ generated iminium on lysine, where site selectivity derives
from a transient imidazolidinone protecting group on the N-
terminus.40 Alternatively, a recent report by Bernardes and co-
workers utilized careful kinetic control with a sulfonyl acrylate
electrophile to target the more nucleophilic lysine residue.41 In
developing a supramolecular bioconjugation strategy, one
important consideration is the feasibility of postmodification
of the functionalized peptide, given the size restrictions of the
host cavity. To this end, we turned our attention to
pentafluorobenzaldehyde (PFBA) as an alternative electrophile
because of its ability to undergo facile SNAr reactions with
thiols after it is appended to the peptide.42−44 We first
subjected lysine and PFBA to aqueous NaCNBH3 conditions
and observed low levels of conversion (<15%) to a mixture of
α-functionalized product 15 and ε-functionalized product 16,
presumably due to the destabilization of the iminium
intermediate by the more electron-deficient arene (see Table
S2 and Figures S34 and S41 in the Supporting Information).
Under host-1 catalyzed reduction conditions, however, >90%
selectivity for ε-functionalized 16 was observed under a range
of basic conditions (pD 10−12) (Figures S34−S38), whereas
+
yield (30%), whereas the NEt4 -blocked host afforded only
trace yields (<5%).
Given the ability of host-1 to maintain reactivity for larger,
partially encapsulated substrates, we next explored lysine as a
potential amphiphilic substrate for site-selective reductive
amination due to the presence of two reactive primary amine
sites: the α-N-terminus as well as the more basic, sterically
accessible ε-terminus. Under host-mediated pD 8 conditions,
however, 4-fluorobenzaldehyde undergoes rapid reduction to
benzyl alcohol 10, leading to low conversions to the reductive
amination product. Increasing the pD of the solution mitigates
this unproductive reaction pathway, and a catalytic loading of
host-1 at pD 12 yields ε-functionalized 12 in 65% yield with
∼3% of α-functionalized product 11 (by 19F NMR, Figure 2a).
Upon addition of a stoichiometric amount of PEt4I (a strongly
binding guest) to host-1, a mixture of products is clearly
observed, with a 28% yield of the ε-functionalized 12 and 15%
yield of 11. Furthermore, control reactions with NaCNBH3 as
the reducing agent conducted in differently buffered solutions
(pD 5−11) consistently yielded a mixture of reductive
amination products, with α-functionalized 11 being the
major product in each case. To further determine whether
host-1 can distinguish between small differences in lipophilicity
and steric accessibility, a competition experiment was
performed with glycine and Nα-Boc-lysine substrates (Figure
2b). Only the lysine-functionalized product 14 was detected
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+
Figure 3. (a) Host-1-mediated reductive amination of peptide 17. (b) LCMS trace of the host-1-mediated reaction, NEt4 -blocked host reaction,
and NaCNBH3 reaction. (c) HRMS of the host-1-mediated reaction. (d) Tandem MS/MS analysis of modified peptide 18.
+
the PEt4 -blocked host afforded low conversions to an almost
1:1 mixture of 15 and 16 (Figures S39 and S40). The site of
modification was further confirmed by subjecting Nα-Boc-
lysine to the host-1-catalyzed conditions followed by Boc
deprotection, which again yielded 16 (Figure S34).
We initially subjected insulin (100 μM) to an excess of
PFBA (20 mM), pyridine−borane (20 mM), and host-1 (20
mM) under basic conditions at room temperature. Analysis of
the crude reaction mixture by mass deconvoluted LCMS
demonstrated high conversion (<90%) to a singly modified
adduct 19, in addition to small amounts of the doubly
modified adduct. Upon blocking the host cavity with
stoichiometric PEt4I to assess background reactivity,48 we
observed only trace amounts of the singly and doubly modified
product, with nearly quantitative recovery of unmodified
insulin, suggesting that the modification was occurring
exclusively within the cavity of host-1. Further optimization
of the reaction conditions revealed that the concentrations of
aldehyde, borane, and host-1 could be lowered without
impacting the conversion or selectivity of the reaction (Figure
4).
To determine the site of modification on 19, the crude
product was subjected to an excess of tris(2-carboxyethyl)-
phosphine (TCEP) to reduce the disulfide bonds present in
the molecule.49 By HRMS, the reduced product mixture
consisted primarily of modified B chain and unmodified A
chain, indicative of lysine modification rather than the glycine
N-terminus (Figure S51). While trace amounts of modified A
chain and unmodified B chain were also detected, this was
largely consistent with the presence of small quantities of the
doubly modified product (resulting from low levels of
background reactivity), as well as unmodified insulin. Finally,
tandem MS/MS analysis of the modified B chain and
unmodified A chain confirmed the site of modification to be
on the desired lysine residue.
We then subjected the 11-amino acid peptide chain 17 with
a single lysine residue and serine N-terminus to the optimized
supramolecular conditions and analyzed the results using an
LCMS-TOF method (Figure 3a). Gratifyingly, 95% conversion
to a major product 18 with a mass corresponding to a single
modification was detected in the presence of host-1 (Figure
3b,c). Blocking the host cavity with NEt4Cl resulted in low
conversion of the starting peptide, indicating that this was a
host-mediated process. Control reactions with NaCNBH3 at
lower pH yielded almost exclusively a single product with the
same m/z as the host-mediated product but with a different
retention time, which was assigned as the N-terminus-modified
product. Modified peptide 18 was sequenced by tandem MS/
MS analysis, and the modification was determined to be
exclusively at the lysine residue, with no N-terminus
modification detected (Figure 3d).
To further assess the scope of this host-mediated reaction,
we next turned our attention to human insulin as a potential
substrate for selective lysine modification. Insulin consists of A
and B peptide chains, containing 21 and 30 amino acids,
respectively, with a single lysine residue in the B chain. In
addition to representing a substantial increase in complexity in
comparison to peptide 17 due to the larger number of amino
acid residues as well as tertiary interactions, insulin contains
two potentially reactive N-termini. While there is precedent for
some selective N-acylation strategies, the lysine-selective
reductive amination of insulin remains relatively rare.45,46
Indeed, under reductive alkylation conditions with sodium
borohydride, Feeney and co-workers observed comparable
levels of modification at both the lysine residue and the glycine
N-terminus.47
CONCLUSIONS
■
In conclusion, we have developed a supramolecular host-
promoted pyridine−borane reduction, which proceeds under
mild, fully aqueous conditions with a substrate scope that
includes enones, ketones, aldehydes, oximes, hydrazones, and
imines. In the presence of catalytic amounts of host-1, lysine
2112
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Wendy Cao − Chemical Sciences Division, Lawrence Berkeley
National Laboratory and Department of Chemistry,
University of California, Berkeley, Berkeley, California
94720, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12479
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Director, Office of Science,
Office of Basic Energy Sciences, and the Division of Chemical
Sciences, Geosciences, and Bioscience of the U.S. Department
of Energy at Lawrence Berkeley National Laboratory (Grant
DE-AC02-05CH11231) and NSF GRFP to W.C. (DGE
1752814). We gratefully thank Dr. Anthony T. Iavarone for
his expertise and guidance in electrospray mass spectrometry
experiments and analysis. We also thank Dr. Hasan Celik and
UC Berkeley’s NMR facility in the College of Chemistry
(CoC-NMR) for spectroscopic assistance. Instruments in the
CoC-NMR are supported in part by NIH S10OD024998.
Figure 4. Host-1-mediated lysine-selective reductive amination of
human insulin under optimized conditions; ESI-TOF data for the
+
host-1-mediated and PEt4 -blocked host reaction.
was site-selectively functionalized at the ε-terminus via
reductive amination with various fluorobenzaldehydes. We
then successfully extended this method to lysine-selective
alkylation of a peptide as well as a protein, demonstrating a
rare example of site-selective supramolecular functionalization
of complex biomolecules. The ability of the reaction to
proceed at micromolar substrate concentrations under fully
aqueous conditions shows promise for future applications of
supramolecular methods in protein bioconjugation.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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■
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Mariko Morimoto − Chemical Sciences Division, Lawrence
Berkeley National Laboratory and Department of Chemistry,
University of California, Berkeley, Berkeley, California
94720, United States
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