Cofactor-Mediated Nucleophilic Substitution Catalyzed by a Self-Assembled Holoenzyme Mimic

Article abstract of DOI:10.1021/acs.joc.9b01880

A self-Assembled Fe₄L₆ cage is capable of co-encapsulating multiple carboxylic acid containing guests in its cavity, and these acids can act as cofactors for cage-catalyzed nucleophilic substitutions. The kinetics of the substitution reaction depend on the size, shape, and binding affinity of each of the components, and small structural changes in guest size can have large effects on the reaction. The host is quite promiscuous and is capable of binding multiple guests with micromolar binding affinities while retaining the ability to effect turnover and catalysis. Substrate binding modes vary widely, from simple 1:1 complexes to 1:2 complexes that can show either negative or positive cooperativity, depending on the guest. The molecularity of the dissociative substitution reaction varies, depending on the electrophile leaving group, acid cofactor, and nucleophile size: small changes in the nature of substrate can have large effects on reaction kinetics, all controlled by selective molecular recognition in the cage interior.

Full text of DOI:10.1021/acs.joc.9b01880

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Article
Cofactor-Mediated Nucleophilic Substitution
Catalyzed by a Self-Assembled Holoenzyme Mimic
Courtney Ngai, Paul M Bogie, Lauren R. Holloway, Phillip C. Dietz, Leonard J. Mueller, and Richard J. Hooley
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01880 • Publication Date (Web): 26 Aug 2019
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The Journal of Organic Chemistry
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Cofactor-Mediated Nucleophilic Substitution Catalyzed by a Self-
Assembled Holoenzyme Mimic
Courtney Ngai , Paul M. Bogie , Lauren R. Holloway, Phillip C. Dietz, Leonard J. Mueller and Richard
J. Hooley*
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Department of Chemistry, University of California-Riverside, Riverside, CA 92521, U.S.A.
Supporting Information Placeholder
4 6
ABSTRACT: A self-assembled Fe L cage is capable of coencapsulating multiple carboxylic acid-containing guests in its
cavity, and these acids can act as cofactors for cage-catalyzed nucleophilic substitutions. The kinetics of the substitution
reaction depend on the size, shape and binding affinity of each of the components, and small structural changes in guest size
can have large effects on the reaction. The host is quite promiscuous, and is capable of binding multiple guests with
micromolar binding affinities, while retaining the ability to effect turnover and catalysis. Substrate binding modes vary
widely, from simple 1:1 complexes to 1:2 complexes that can show either negative or positive cooperativity, depending on
the guest. The molecularity of the dissociative substitution reaction varies, depending on the electrophile leaving group, acid
cofactor and nucleophile size: small changes in the nature of substrate can have large effects on reaction kinetics, all controlled
by selective molecular recognition in the cage interior.
of “cofactor-mediated catalysis” with synthetic receptors,
namely the use of a host:guest complex to catalyze reaction
between additional reactants bound inside the parent host.
INTRODUCTION
The scope of enzymatic reactions is widely enhanced by
the use of cofactors. Species such as flavins, pyridoxal
1
2
One strategy is to use a very small cofactor, namely a
3
4
phosphate (PLP) and cobalamin are bound by their
respective apoenzymes to form a holoenzyme complex that
is capable of binding additional substrates, mediating their
reactivity. The mechanism of action of biological cofactors
has inspired many famous synthetic transformations over
+
-
14
4 6
solvent-coordinated H or OH ion. Alternatively, M L
catecholate hosts in water can bind organometallic
15
species and can effect small molecule transformations
such as intermolecular cyclizations and isomerizations,
1
6
among others.
Larger cofactors usually require
5
the years.
1
7
supercapsules such Pd12
self-assembled resorcinarene hexamers, which have
interior cavity volumes of greater than 1375 Å . This
L
24 and Pd24
L
48 nanospheres, or
While synthetic chemists are inspired by the innate
mechanisms of cofactor-mediated catalysis, the molecular
recognition aspects inspire supramolecular chemists. This
can motivate multiple avenues of research: external
cofactors can be used to switch catalyst function or as
allosteric effectors in a wide range of catalytic processes.
18
3 19
6
allows the binding of multiple small molecules in internal
“
nanophases”, and have been used to promote either
20
21
Brønsted acid or gold catalyzed cyclization reactions,
iminium-catalyzed conjugate additions, and carbonyl-
olefin metatheses. Other examples of hosts that can
7
22
Alternatively, a small molecule cofactor can be bound
internally in the host cavity, which then promotes a reaction
between other species also bound in that site. This could be
defined as “holoenzyme”-mimicry, in that host active site
mediates the reaction of a bound cofactor (such as PLP,
flavin, etc.), enhancing rate and providing stereoselectivity.
This requires binding multiple different species in a
23
exploit cofactor effects are metalloporphyrin assemblies,
which use ligand to the metal centers to control selectivity
24
and rate in processes such as hydroformylation.
One of the advantages of smaller, more defined host
structures is that the size of the individual components can
be varied to affect the reaction outcome: by changing the
size and shape of the cofactor, different selectivities could
be observed for different reactants. Smaller hosts can have
their own issues in supramolecular catalysis, however, most
8
synthetic host, as well as activating the substrates and
9
turning them over, which is still a significant challenge for
synthetic host species. Coencapsulation of two or more
guests to form homoternary complexes is relatively well
2
5
notably product inhibition and poor turnover. Here we
show that an organic-soluble metal-ligand cage complex can
act as a host environment for cofactor-mediated catalysis.
The cage is a promiscuous, yet high affinity host, and
multiple guests can be bound, reacted and released. The
reaction kinetics depend on the molecular recognition of all
the components in the reaction, and small changes in
1
0
known, but formation of heteroternary complexes is
1
1
rarer. Additionally, most of these examples exhibit tight
host:guest binding to allow coencapsulation, so turnover
can be problematic, limiting their use as catalysts. Many
supramolecular catalysts either promote unimolecular
1
2
rearrangements,
or promote the dimerization of
1
3
complementary substrates. There are far fewer examples
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substrate structure can have large effects on the host-
catalyzed reaction.
Triphenylmethanol 4a was heated with 1.25 mol.-eq. n-
propanethiol in the presence of 5% cage 1 and 30% cofactor
3a in CD CN, and the initial rate of the reaction forming
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RESULTS AND DISCUSSION
thioether 5a was monitored by H NMR (Figure 2).
Interestingly, the combination of 1 and 3a is an effective
catalyst for the reaction, showing a >50-fold increase in
initial rate when compared to the same concentration of 3a
in the absence of 1. The process is not catalyzed by cage 1 in
the absence of catalyst at all. The rate of the cofactor-
mediated process with 1•3a is ~30 times slower than the
We recently synthesized the large tetrahedral Fe
complexes 1 and 2 (Figure 1). Acid-functionalized cage 2
4
L
6
cage
26
is an effective biomimetic catalyst, capable of catalyzing
26
sequential
tandem
reactions
and
nucleophilic
substitutions such as the thioetherification of
2
7
triphenylmethanol. This process involves the formation of
ternary host:guest complexes, and hints at the possibility of
cofactor-mediated catalysis in synthetic receptors. As the
thioetherification of triphenylmethanol 4a with
alkylmercaptans is well-suited for mechanistic analysis in
these cage complexes, we initially tested whether
unfunctionalized cage 1 could promote the reaction in the
presence of a suitably sized acidic cofactor.
2
7
reaction catalyzed by 5% acid-functionalized cage 2, as
might be expected, but this initial experiment illustrates
that the presence of cage 1 can significantly enhance the
activity of the free acid catalyst, despite the fact it has no
reactive functional groups. This suggests that molecular
recognition effects are involved, and the acid is indeed
acting as a “cofactor”, and the cage as a holoenzyme mimic.
Importantly, cage 1 is stable to the presence of acid 3a, and
no decomposition is seen during the reaction, even after 12
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h at reflux in CD CN (Supporting Figure S4). It is intolerant
to stronger acids (e.g. camphorsulfonic acid (CSA) or
2
6
3 2
CF CO H ) at high temperatures, however. Rapid
decomposition and solvolysis of the iminopyridine groups
is seen in the presence of 6 eq. CSA after 5 mins at 80 °C in
CD CN.
3
Figure 2. Cofactor-mediated catalysis with cage 1 and acid
a. Reaction progress over time for the thioetherification of
3
electrophile 4a with PrSH and either 5% cage 2, 5% cage
1/30% 3a, or 30% 3a alone as catalyst. [4a] = 15.8 mM, [PrSH]
=
19.8 mM, reactions were performed at 80 °C in CD
3
CN.
To determine whether the accelerated reaction with 1•3a
was due to molecular recognition, we investigated the guest
binding properties of cage 1 in more detail. We have
previously shown that these extended fluorenyl cages,
notably acid-functionalized cage 2, show strong binding
-1
affinities (up to 200,000 M ) for small molecules in
2
6,27
acetonitrile.
Unfunctionalized cage 1 has a substantially
larger cavity than acid cage 2, however, and cannot exploit
polar interactions between the host COOH groups and
guest. In addition, the lack of bulky acid groups creates
larger “gaps” between the walls of the cage (Figures 1b, 1c),
which should lower guest affinity, especially for small
neutral species.
Figure 1. a) Structures of Fe
2; minimized structures of the S isomers of b) cage 1; c) cage
4
L
6
cage 1 and acid-decorated cage
4
Analysis of the host properties of cage 1 is not trivial. The
2
6
3
interior cavity of 1 is large (~600 Å ), and all of the
2 (SPARTAN, Hartree-Fock); d) structures of the acid cofactors;
e) summary of the acid catalyzed substitution processes tested
components are small enough to theoretically form ternary
(
or in some cases higher) complexes with 1. The gaps
(
1•(3a-e) = 1:6 ratio of cage: cofactor).
between the ligand walls are also large, and all guests tested
show fast in/out exchange rates on the NMR timescale.
Chemical shift changes of protons in either the guest or the
The initial tests were performed with the fluorene-based
diacid 3a, a direct synthetic precursor to acid cage 2.
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host in H NMR experiments are small, and the fact that cage
Acid 3d
Acid 3e
25.5 ± 1.0
(OctS)2
76.1 ± 3.8
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1 exists as a mixture of three metal centered isomers in
solution (48% C3, 41% S
complexity. The high freezing point of CD
2.40 ± 0.15
2
6
4
, 11% T) only adds to the
CN limits low
a
3
in CH CN, [1] = 1.5 μM, absorbance changes measured at
3
2
8
3
00/330nm and 370 nm.
temperature investigations, and the exchange rates are too
fast to allow effective NOE buildup in 2D NMR experiments.
Fortunately, UV/Vis absorbance titrations are an effective
method of investigating the recognition events. The binding
constants are high enough that strong changes in
absorbance of cage
concentrations in CH CN. Each guest was titrated into a 1.5
μM solution of 1 in CH CN, and the changes in absorbance at
The calculated binding affinities are all strong, with the
weakest affinity shown by pivalic acid 3e. Every other guest
has an affinity of >10 M , which corresponds to >95%
occupancy at millimolar concentrations, so competitive
guest binding effects are clearly relevant in any catalytic
process. The larger guests show greater affinities, as might
be expected, and anthroic/naphthoic acids 3b and 3c are
4
-1
1 occur at even micromolar
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both 330 and 370 nm were recorded and analyzed. The
-1
very strongly bound, with affinities of ~100,000 M .
binding isotherms were fit with both 1:1 and 1:2 models, 28
Notably, the thioethers 5a and 5b are strongly bound as
well, indicating that product inhibition is a factor that must
be considered in any cage-catalyzed reactions with 1.
Unfortunately, the complex fitting equations prevent
unambiguous proof of 1:2 heterocomplexes with multiple
different guests. Titration of 3a into 1•PrSH shows
additional changes in absorbance, but is not possible to
determine whether this is due to expulsion of PrSH, or
formation of heteroternary complexes.
and we then analyzed the best fit for each guest. The results
are summarized in Table 1: for the full fitting details,
including fitting curves, variances and error analysis, see
Supporting Information.
Twelve different components (Figure 1d) were analyzed
that would allow a range of mechanistic investigations into
the thioetherification reaction. They consisted of two trityl
electrophiles 4a and 4b, two different sized nucleophiles n-
propanethiol (PrSH) and n-octanethiol (OctSH), five acidic
cofactors 3a-e, as well as the thioether products 5a and 5b,
2
and dioctyldisulfide (OctS) . All of the components show
strong affinity for the cage, interestingly, even small species
such as PrSH. In each case, the binding isotherms were fit to
both the 1:1 and unbiased 1:2 binding models and the
variances calculated. The significance of the 1:2 model was
judged based on the inverse ratio of the squared residuals
compared to the 1:1 model, and quantified via p-value.
Three general patterns emerged from this analysis, and
these are summarized in Table 1 (and Tables S2 - S4). Three
guests unambiguously showed best fit to the 1:2 binding
model, with p-values below 0.001, and are labeled as the 1:2
substrates in Table 1: OctSH, trityl ether 3b and cofactor 3a.
In these cases, two equilibrium constants were extracted,
defined as K
:1 and 1:2 host:guest complexes.
Table 1. Binding Affinities of Reaction Components in Cage
1 2
and K , illustrating the sequential formation of
1
a
1.
Figure 3. Minimized structures (SPARTAN, Hartree-Fock) of a)
S
4 2 4 2 4 4
-1•3a ; b) S -1•4a ; c) S -1•3b; and d) S -1•3b•4a•PrSH.
3
-1
3
-1
1:2
K
1
x 10 M
K
2
x 10 M
α
)28a
The substrates that form 1:2 complexes are especially
interesting. As the 1:2 binding model was unbiased, the
cooperativity of the binding process was not assumed in the
Substrate
2
(4K /K
1
OctSH
174 ± 43
0.78 ± 0.53
0.018
Ether 4b
Acid 3a
47.1 ± 8.5
19.0 ± 11
2.11 ± 0.38
244 ± 89
0.18
51
2 1
model, and the cooperativity factor α (defined as 4K /K )
2
8a
can be analyzed. Interestingly, the cooperativity of the 1:2
substrates is not constant. While OctSH and ether 3b show
negative cooperativity (α <1), diacid cofactor 3a shows
strong positive cooperativity, with α = 51. This is
presumably due to self-complementary hydrogen bonds
between the two diacids, but why this is not seen for the
other acids 3b-3e is not clear. Molecular modeling sheds
some light on the binding modes. The large guests fill the
space on the interior quite effectively in a 1:2 manner: the
3
-1
3
-1
1:1
K
a
x 10 M
K
a
x 10 M
Substrate
PrSH
58.5 ± 4.7
95.4 ± 5.5
Alcohol 4a
Thioether
14.5 ± 0.77
24.8 ± 1.5
Acid 3b
5a
Acid 3c
102 ± 5.2
Thioether
91.7 ± 7.8
5b
minimized structures of 1•3a
2 2
and 1•4b (SPARTAN,
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Hartree-Fock) are shown in Figure 3a and 3b. The cavity is Having illustrated the binding affinity of the various
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easily spacious enough to occupy two guests, and the
relatively large exit/entry portals can allow fast guest
exchange. The cavity is even large enough to conceivably
form a quaternary complex with all three reactants (Figure
components, we investigated the effect of the cage on the
kinetics of the various acid-catalyzed thioetherification
processes. The components of the reaction were
systematically varied, focusing on small changes in
component structure that should have minimal effects on
the reaction in the absence of cage. The two electrophiles
triphenylmethanol 4a and its ethyl ether 4b have similar
reactivities and only small differences in size. The five
different acid cofactors (3a-e, Figure 1) were chosen such
that the size of the cofactor could be varied significantly,
while retaining relatively similar acidities. The inspiration
for the process, diacid 3a, is the largest substrate, and has a
pKa of ~3.7 (based on comparison with 3,3-
3
d), although this would have substantial entropic
penalties. This of course introduces the question of why
there is observable affinity for all the guests, and at such
high binding constants, even for small guests such as PrSH.
The 1•3b complex in Figure 3c illustrates the large spaces
in the cavity upon binding only one guest. Obviously the
remainder of the cavity can be filled by solvent molecules,
0
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29
but Rebek’s 55% occupancy rule is not dominant here. The
most reasonable suggestion is that the small, polar guests
interact with the octacationic cage and its aromatic walls via
CH-π and π-π interactions, and these interactions allow
transient formation of host:guest complexes. This is not
unprecedented: the Nitschke lab has shown that a variety of
Fe-iminopyridine cages with large cavities can show rapid
31
dimethylglutarate ). The other cofactors vary slightly in
3
1
pKa (3b = 3.65, 3c = 3.69, 3d = 4.20, 3e = 5.03), but have
3
3
substantial differences in volume (3a = 244 Å , 3b = 159 Å ,
3
3
3
3c = 122 Å , 3d = 96 Å , 3e = 84 Å ). Finally, the two
nucleophiles PrSH and OctSH show highly similar
nucleophilicity but significantly different overall size, with
3
0
in/out kinetics with small molecule guests, and only when
the exit portals are reduced in size do kinetically stable
Michaelis complexes form. It is important to note that
accurate structural information about where the guests
reside complexes is still lacking, due to the limited
information available from NMR analysis. These cages have
3
3
volumes of 68 Å and 136 Å , respectively.
The first tests were to determine the effect of varying the
cofactor catalyst, keeping the nucleophile and electrophile
constant (alcohol 4a and PrSH, respectively). The ratio of
cage:cofactor was kept constant at 5% cage 1 and 30%
1
5a, 30
no large flat panels creating a box-like enclosure,
rather
3
cofactor 3a-3e, with [4a] = 15.8 mM in CD CN. This 1:6 ratio
the walls are very much edge-oriented and so the usual
of cage to cofactor will be described as 1•3a-e for the rest
of this paper. The reactions were run to ~25% completion
to ensure accuracy in initial rate measurement (although
some of the faster reactions proceeded further in the same
timeframe). The initial rates for the cage-mediated
processes (V(1•3a-e)) and the background rate with 30%
cofactor in the absence of cage (V(3a-e)) are shown in Table
2 and Figure 4. The different cofactors show quite different
catalytic activities, even in the absence of cage. The reaction
rates catalyzed by “free” cofactors 3a-3e vary somewhat,
but they do not follow the trend of pKa; naphthoic acid 3c is
the best catalyst, and diacid 3a is by far the worst, despite
their similar pKa. The relative order of effectiveness is
30a
definition of guests being “inside” or “outside” the cage is
less clear. The models in Figure are plausible
3
representations of host:guest complexes, but are not the
only possibilities that would allow promoted reaction. What
is clear from the binding studies is that the host brings
multiple species into close proximity, which allows
accelerated reactions.
3
c>3d>3b>>3e>3a. None of the free catalysts 3a-3e are
particularly effective, however, with all of the reactions only
reaching <30% conversion at best after 6h reflux. In each
case, the reactions were very clean: the only observed
species in the NMR were the reactants, thioether products
and a small amount of disulfide (see below) in certain cases.
No ester byproducts from tritylation of the acids were seen,
either in the control or cage-catalyzed examples.
Table 2. Supramolecular Cofactor-Mediated Catalysis.a
Acid
cofactor x10-4 mM/min x10-4 mM/min
V(1•(3a-e)),
V(3a-e),
V(1•3(a-e))/
V(3a-e)
3a
39
229
126
109
92
0.7
19
67
33
8
56
12
1.9
3.3
12
Figure 4. Dependence on cofactor size. Reaction progress
over time for the thioetherification of electrophile 4a and 4b
with PrSH a) 5% cage 1/30% cofactor 3a-e catalyst, and b)
3b
3c
3
8
0% 3a-e alone. [4a] = 15.8 mM, reactions were performed at
0 °C in CD CN.
3d
3
3e
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a
[4a] = 15.8 mM, [RSH] = 19.8 mM, reactions were performed
1
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at 80 °C in CD
CN. Initial rates were determined using the first
3
set of linear timepoints under 50 % conversion by comparing
Δ[5a]/t(min). Concentrations were confirmed using dioxane as
a standard (7.9 mM).
When 5% cage 1 is added, the relative rates of reaction
change markedly, and the rate acceleration due to the
presence of catalytic cage 1 varies significantly with the
nature of the acid cofactor. The overall reaction rate order
is 1•3b>1•3c~1•3d>1•3e>1•3a. Addition of cage 1 has the
largest effect on the reactions catalyzed by diacid 3a,
anthroic acid 3b and pivalic acid 3e, with each complex
showing at least a 10-50 fold enhancement in initial rate
compared to that with the free acid. In contrast, the
reactions catalyzed by naphthoic acid 3c and benzoic acid
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d are only accelerated ~2-fold by the presence of 5% cage
1. In addition, simply varying the cofactor in the cage-
mediated process from 3a and 3b causes a 15-fold rate
difference, despite the fact that the cofactor pKas are
essentially the same and all other conditions are identical.
The thioetherification process caused no decomposition of
the cage (Figure S4), even under extended reaction times,
but some oxidative dimerization of the PrSH nucleophile
was observed in the slower reactions, presumably caused
by small amounts of free Fe leached from the cage and
atmospheric oxygen. This reaction was slower than the
thioetherification reaction, and only small amounts of
Figure 5. Reaction dependence on cofactor concentration.
a) Reaction progress over time with varying [3a]; b) reaction
rate vs [3a]; c) Reaction progress over time with varying [3d];
d) reaction rate vs [3b]. [4a] = 15.8 mM, [PrSH] = 19.8 mM,
II
reactions were performed at 80 °C in CD CN.
3
The relevant questions are whether the reaction rate is
dependent on the concentration of cofactor and/or
nucleophile, and how this dependence changes upon
varying the nature of the electrophile between alcohol 4a
and ether 4b. The reaction rate is indeed dependent on
(
2
PrS) were observed. Interestingly, this small amount of
free Lewis acid is not capable of catalyzing the
thioetherification: no reaction was observed after extensive
heating with 1 alone.
[cofactor], as might be expected - Figure 5 shows the
The next steps were to investigate which components
were directly involved in the rate equation: while the
thioetherification reaction with “free” catalyst is an S 1
N
variation in initial rate upon varying [3a] or [3b] from 1.6
mM to 4.8 mM (10 to 30% with respect to electrophile)
while keeping the [1] constant at 15.8 mM, and the reaction
rate increases with increasing [3b]. The observations are
somewhat surprising: the rate of the acid-catalyzed reaction
is not affected by variations in concentration of diacid 3a.
The acid must be involved in the reaction, as the process
does not occur without it, nor can it be catalyzed by cage 1
in the absence of acid. The explanation lies in the unusual
binding characteristics of diacid 3a: as the binding is
strongly positively cooperative (α = 51), the resting state is
process and will have no dependence on [nucleophile],
introducing the cage 1 host into the reaction will change
this. If the cofactor, electrophile and/or nucleophile are
bound by the cage before the rate determining step, the
reaction rate will show a dependence on [nucleophile]. We
therefore performed initial rate studies with varying
electrophile type (4a or 4b), varying [cofactor] and varying
concentration and size of nucleophile (Figures 5 and 6). For
simplicity, we narrowed down the focus to the cofactors
that were most strongly affected by the presence of cage 1,
diacid 3a and anthroic acid 3b.
1•3a , not 1•3a. As the binding is so high, even at 1:1
2
cage:guest ratio, the inactive 1•3a dominates the resting
2
state, so the rate is essentially independent of 3a. In
contrast, anthroic acid 3b, which binds in a 1:1 manner,
shows saturation kinetics, with rate increasing with
increasing [3b], but slowing at high [3b]. This is likely due
to inhibition by saturating the cage with excess cofactor 3b.
This reactivity profile indicates the possibility of forming
1
•3b
2
, as was hinted at by the fitting analysis. If a small
can form, it is not positively cooperative,
amount of 1•3b
2
and the resting and active states of the cage:cofactor
complex are identical.
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Page 6 of 10
conditions, the observed rate was slightly faster at 79 x10-4
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mM/min. However, upon changing the concentration of
PrSH, the rate of reaction of ether 4b remains identical,
whereas that with alcohol 4a increases significantly with
increasing [PrSH]. This variation in dependence on
nucleophile concentration is not due to differing
mechanisms of reaction between 4a and 4b in the absence
2
7
of cage: using either strong acids such as CF
catalyst shows no change in rate with varying [PrSH], as
would be expected for an S 1 reaction. The structural
3
CO
2
H
as
N
change in electrophile is small - there is a difference in
basicity between 4a and 4b (conjugated acid pKa of ~-3.5
vs -2), as well as a small difference in size, but the cation
formed upon reaction is identical, so the change in
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[
nucleophile] dependency is unusual. This observation
2
7
mirrors the effect seen with acid-functionalized cage 2,
where molecular recognition effects change the
molecularity of the reaction. In this case, similar changes in
nucleophile dependence are observed for a cofactor-
mediated process.
When anthroic acid 3b is used as cofactor, the kinetic
behavior of the reaction changes significantly. The rate of
reaction of alcohol 4a with PrSH catalyzed by 1•3b is much
-4
faster (260 x 10 mM/min) than with 1•3a, whereas the
reaction rate with ether 4b is essentially unchanged (70 x
-4
1
0 mM/min). In both cases catalyzed by 1•3b, there is no
dependence on [PrSH]. Finally, the nature of the
nucleophile was varied, and the larger n-octanethiol
(
OctSH) was used in place of PrSH. Figures 6e and 6f show
the rate profiles for the reaction of OctSH with electrophiles
a and 4b, with 1•3a as catalyst. The initial rates of
thioetherification are faster than those with PrSH (k(4a) =
4
-4
-4
1
35 x 10 mM/min, k(4b) = 150 x10 mM/min with 1.25
eq. OctSH). The dependence on nucleophile concentration
is similar to that shown by PrSH: ether 4b has no
dependence on [OctSH], whereas alcohol 4a does.
Figure 6. Reaction dependence on nucleophile
concentration and size. Reaction progress over time with a)
4a, varying [PrSH], 1•3a catalyst; b) 4b, varying [PrSH], 1•3a
catalyst; c) 4a, varying [PrSH], 1•3b catalyst; d) 4b, varying
[PrSH], 1•3b catalyst; e) 4a, varying [OctSH], 1•3a catalyst; f)
4b, varying [OctSH], 1•3a catalyst. [4a, 4b] = 15.8 mM, [1] =
As well as the differences in thioetherification rate, the
reaction with OctSH displayed one other notable difference
from that with PrSH: OctSH is oxidatively dimerized to the
0
.8 mM, [3a,3b] = 4.8 mM. Reactions were performed at 80 °C
in CD CN.
The other unusual observation is that the putatively “S
disulfide (OctS) by cage 1 at a much faster rate. We have
2
3
previously observed that more reactive aryl thiols can be
2
7
N
1”
oxidized to the disulfides by Fe-containing cages, but
oxidation of alkyl thiols is very sluggish. Despite the two
thiols having highly similar oxidation potential, OctSH was
oxidized by 5% cage 1 at a rate 4-fold faster than PrSH.
reaction to form thioether 5a shows variable rate
dependences when the components are varied, including
showing rate dependence on the concentration of
nucleophile. When small molecules are used to catalyze this
The presence of the cage has a variety of effects on the
reactions, some subtle, and some that are quite remarkable.
Figure 7 shows a summary of some of the effects of the cage
on the reaction process. Not all of the possible equilibria are
shown, for clarity - as there are as many as 4 components in
the reaction mixture as well as the cage, and as some of
them can form 1:1 and 1:2 homo- and hetero-ternary
complexes, there are many possible host:guest processes
occurring during the reaction. Despite the host showing
strong affinity for all components of the reaction, the rapid
in/out exchange rates of the substrates allow the cofactor-
mediated catalysis to be successful.
2
7
reaction, no rate dependence on nucleophile is seen: only
when cage catalysts capable of molecular recognition (such
as 2) are used. Figure 6 shows the initial rates observed for
the cage-catalyzed thioetherification reaction at varying
concentrations of nucleophile. The six entries in Figure 6
show these effects on reactions between electrophiles 4a
and 4b, with PrSH and OctSH nucleophiles, and with
cofactors 3a and 3b. Even at first glance, it is obvious that
small changes in reactant structure effect large changes in
rate and dependence on [nucleophile] in the cage-catalyzed
reaction.
Figure 6a clearly shows that the rate of reaction between
The general accelerated cofactor-mediated process is
illustrated in Figure 7a, covering the reactions that do not
show nucleophile dependence (e.g. with 4b, 3b, etc.). In this
4a and PrSH catalyzed by the 1•3a complex is dependent
-4
on [PrSH]. The rate at [PrSH] = 19.8 mM was 39 x10
mM/min. When ether 4b was subjected to the same
N
case, a standard S 1 mechanism is occurring, and cation
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The Journal of Organic Chemistry
solvent molecules from the cavity. Other arguments could
formation is the rate determining step. The electrophile and
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cofactor can each bind in the cavity of 1, and the accelerated
reaction occurs when the electrophile 4 is activated by the
1•3 complex. The rate acceleration is controlled by the
relative proportion of the 1•3•4 complex in solution. This is
not dependent on the affinity of the individual components:
for example, naphthoic acid 3c has essentially the same
affinity for 1 as anthroic acid 3b, but gives only a 2-fold
acceleration of the 4a/PrSH thioetherification is seen, as
opposed to a 12-fold acceleration with 3b. The strongest
accelerations are seen with reactants that show synergistic
co-encapsulation in the host cavity. It should be noted that
the products 5a/5b have stronger affinity for 1 than the
reactants, and some product inhibition is observed at high
conversions, in contrast with acid cage 2, where the
be made for pre-equilibrium binding of nucleophile in 1
affecting the rate, but as all other combinations show no
nucleophile dependence, this is unlikely. The oddity is that
the combination of 3a and 4a is unique – only in this case is
nucleophile dependence seen, and this combination shows
a much larger rate acceleration than with the other
cofactors. The most likely reason is that the effects causing
2
the positive cooperativity in formation of 1•3a (self-
complementary H-bonding with the diacid) also favor the
formation of heteroternary complexes with the alcohol
electrophile, and can contribute to binding the nucleophile
too. This phenomenon does require further investigation,
however.
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Finally, the competing oxidative dimerization of the
nucleophile is an interesting illustration of the favorable 1:2
binding of the longer OctSH in cage 1 (Figure 7c). The
accelerated dimerization of OctSH can be easily explained
by the colocalization of the two thiols in the cage interior,
2
7
products has a lower affinity than the reactants.
II
with the reaction promoted by small amounts of free Fe
salts. PrSH is smaller and does not favor 1:2 complexes,
hence the dimerization rate is slower.
CONCLUSIONS
4 6
In conclusion, we have shown that a self-assembled Fe L
cage is capable of coencapsulating multiple carboxylic acid-
containing guests in its cavity, and these acids can act as
cofactors for cage-catalyzed nucleophilic substitutions. The
most important observations are the non-linear
dependency of the reaction on cofactor concentration, the
differing rate accelerations for differently sized cofactors
and the variable dependency of the reaction nucleophile
concentration. These observations illustrate that molecular
recognition of one or more reaction components is key to
the reaction outcomes. Small changes in the size and shape
of the reactants and catalysts can have large effects on the
reaction profile, in unexpected ways. Differently sized
cofactors, nucleophiles and electrophiles all affect the
reaction rate and molecularity differently, even when they
have similar reactive properties outside the cage.
Figure 7. Mechanistic possibilities in the cofactor-
mediated process. a) “Standard” cofactor-mediated process;
b) requirements for nucleophile-dependent kinetics; c)
accelerated dimerization of large nucleophiles by favorable
ternary complex formation.
EXPERIMENTAL
The most unusual reactivity is shown by the combination
of cofactor 3a and electrophile 4a (Figure 7b). Whereas all
General Information. Cages 1 and 2, and cofactor 3a
26
other combinations showed S
N
1-type kinetics, with the cage
were synthesized according to literature procedures. See
1
13
controlling the overall rate, using diacid 3a as cofactor with
triphenylmethanol showed a rate independent of cofactor
concentration, as well as dependent on nucleophile
concentration. As discussed previously, the unique positive
that publication for full characterization. H, and C spectra
were recorded on Bruker Avance NEO 400 MHz or Bruker
Avance 600 MHz NMR spectrometer. The spectrometers
were automatically tuned and matched to the correct
1
13
cooperativity in forming the 1•3a
2
complex can explain the
operating frequencies. Proton ( H) and carbon ( C)
chemical shifts are reported in parts per million () with
respect to tetramethylsilane (TMS, =0), and referenced
internally with respect to the protio solvent impurity for
lack of dependence on cofactor concentration with 3a. The
reasons for dependence on [nucleophile] are less obvious.
With acid-bearing cage 2, strong dependence on
2
7
1
13
[
nucleophile] was observed, but that only requires
CD CN ( H: 1.94 ppm, C: 118.3 ppm). Deuterated NMR
3
formation of ternary host:guest complexes. For the
cofactor-mediated process with cage 1, introducing
nucleophile before the rate-determining step would require
solvents were obtained from Cambridge Isotope
Laboratories, Inc., Andover, MA, and used without further
purification. Spectra were digitally processed (phase and
baseline corrections, integration, peak analysis) using
Bruker Topspin 1.3 and MestreNova. All other materials
were obtained from Aldrich Chemical Company (St. Louis,
MO), or Fisher Scientific (Fairlawn, NJ), and were used as
received. Solvents were dried through a commercial solvent
the formation, however briefly, of
•3a•4a•PrSH complex. The molecular modeling in Figure
d suggests that this is plausible, as all three components
a
quaternary
1
3
can fit in the cavity of 1. The entropic penalty of forming a
quaternary complex could be overcome by expulsion of
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purification system (Pure Process Technologies, Inc.).
Corresponding Authors
Page 8 of 10
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UV/Vis spectroscopy was performed on a Cary 60
Photospectrometer using the Varian Scans program to
collect data.
*
E-mail: richard.hooley@ucr.edu
These authors contributed equally to the manuscript.
a
Synthesis of octyl trityl sulfide 5b. Trityl chloride (100
mg, 0.36 mmol) was placed in a Schlenk flask with a stir bar
ACKNOWLEDGMENTS
The authors would like to thank the National Science
Foundation (CHE-1708019 to R.J.H., CHE-1710671 to L.J.M.,
and CHE-1626673 for the purchase of Bruker NEO 600 and
NEO 400 NMR spectrometers) for funding.
2
and purged with N . n-Octanethiol (0.12 ml, 1.8 mmol) was
added to the flask, and the reaction was stirred at 80 °C in a
heating mantle for 12 h. The solvent was removed and the
product dried in vacuo to yield pure product as a white
1
crystalline solid (105.6 mg, 76 %). H NMR (400 MHz,
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0
1
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3
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0
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found 387.2141 ([M-H] ).
General procedure for substitution reactions.
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internal standard (0.5 mol. -eq., 3.2 μmol, 1.6 μL of 2 M
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CN) or 30% acid 3 alone. The
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3
1
shaken to dissolve all solids. An initial H NMR spectrum of
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0 °C and the reaction progress monitored over time. Rate
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1
.5 µM solution of cage 1 was prepared in spectroscopic
grade CH CN via dilutions from a 0.3 mM stock solution, and
added to a UV-Vis cuvette. To this solution was then added
µL aliquots from a 4.5 mM solution of the corresponding
5
341–5370.
3
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1
guest molecule, equating to one molar equivalent guest to
cage. These additions were continued until there was no
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using the Nelder-Mead method from the change in
absorbance at two points (300nm/330nm and 370nm), the
data was fit to either a 1:1 or 1:2 binding model and the
variance used to determine best fit using a non-linear least-
1
508.
(
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Acad. Sci. U.S.A. 2018, 115, 9379–9384.
squares (maximum likelihood) approach written within the
Mathematica programming environment.28 See Supporting
Information for equations and full description of the fitting.
ASSOCIATED CONTENT
Supporting Information
Binding analysis including spectroscopic data and binding
isotherms, kinetic data and computational analysis of curve
fittings. This material is available free of charge via the Internet
at http://pubs.acs.org.
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1 ) (a) Kang, J. M.; Rebek, J., Jr. Acceleration of a Diels-Alder reaction
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Aqueous hydrogen bonds in hydrophobic cavities. J. Am. Chem.
Soc. 2010, 132, 7194–7201. (d) Mahata, K.; Frischmann, P. D.;
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Würthner, F. Giant electroactive M L tetrahedral host self-
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