Enzyme-mediated dynamic combinatorial chemistry allows out-of-equilibrium template-directed synthesis of macrocyclic oligosaccharides

Article abstract of DOI:10.1039/c9sc03983j

We show that the outcome of enzymatic reactions can be manipulated and controlled by using artificial template molecules to direct the self-assembly of specific products in an enzyme-mediated dynamic system. Specifically, we utilize a glycosyltransferase to generate a complex dynamic mixture of interconverting linear and macrocyclic α-1,4-d-glucans (cyclodextrins). We find that the native cyclodextrins (α, β and γ) are formed out-of-equilibrium as part of a kinetically trapped subsystem, that surprisingly operates transiently like a Dynamic Combinatorial Library (DCL) under thermodynamic control. By addition of different templates, we can promote the synthesis of each of the native cyclodextrins with 89-99% selectivity, or alternatively, we can amplify the synthesis of unusual large-ring cyclodextrins (δ and ?) with 9 and 10 glucose units per macrocycle. In the absence of templates, the transient DCL lasts less than a day, and cyclodextrins convert rapidly to short maltooligosaccharides. Templates stabilize the kinetically trapped subsystem enabling robust selective synthesis of cyclodextrins, as demonstrated by the high-yielding sequential interconversion of cyclodextrins in a single reaction vessel. Our results show that given the right balance between thermodynamic and kinetic control, templates can direct out-of-equilibrium self-assembly, and be used to manipulate enzymatic transformations to favor specific and/or alternative products to those selected in Nature.

Full text of DOI:10.1039/c9sc03983j

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Enzyme-mediated dynamic combinatorial chemistry
allows out-of-equilibrium template-directed
synthesis of macrocyclic oligosaccharides†
Cite this: Chem. Sci., 2019, 10, 9981
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
*
Dennis Larsen
and Sophie R. Beeren
We show that the outcome of enzymatic reactions can be manipulated and controlled by using artificial
template molecules to direct the self-assembly of specific products in an enzyme-mediated dynamic
system. Specifically, we utilize
a glycosyltransferase to generate a complex dynamic mixture of
interconverting linear and macrocyclic a-1,4-D-glucans (cyclodextrins). We find that the native
cyclodextrins (a, b and g) are formed out-of-equilibrium as part of a kinetically trapped subsystem, that
surprisingly operates transiently like a Dynamic Combinatorial Library (DCL) under thermodynamic
control. By addition of different templates, we can promote the synthesis of each of the native
cyclodextrins with 89–99% selectivity, or alternatively, we can amplify the synthesis of unusual large-ring
cyclodextrins (d and 3) with 9 and 10 glucose units per macrocycle. In the absence of templates, the
transient DCL lasts less than a day, and cyclodextrins convert rapidly to short maltooligosaccharides.
Templates stabilize the kinetically trapped subsystem enabling robust selective synthesis of cyclodextrins,
as demonstrated by the high-yielding sequential interconversion of cyclodextrins in a single reaction
vessel. Our results show that given the right balance between thermodynamic and kinetic control,
templates can direct out-of-equilibrium self-assembly, and be used to manipulate enzymatic
transformations to favor specific and/or alternative products to those selected in Nature.
Received 9th August 2019
Accepted 2nd September 2019
DOI: 10.1039/c9sc03983j
rsc.li/chemical-science
system (Fig. 1). We show how molecular recognition of arti-
cial templates can be exploited to obtain alternative prod-
Introduction
ucts to those selected by Nature.
Biomolecular templates dene the precise outcomes of
enzyme-catalyzed reactions in essential biological processes,
such as DNA replication, translation and transcription.
Inspired by these, and other template-driven processes in
biology, chemists have exploited templates to control the
chemical synthesis of complex architectures, such as macro-
cycles,1 catenanes,2 cages3 and knots.4 Dynamic combinato-
rial chemistry (DCC), in particular, has emerged as a powerful
tool to direct the molecular self-assembly of oligomers
whereby monomers are linked together using reversible
reactions, and template molecules bind to, stabilize, and
amplify specic targeted oligomers.1,4,5 To date, DCC has
remained primarily in the realm of synthetic organic chem-
istry with only a few examples reported of the use of reversible
enzyme-mediated reactions to generate relatively small
dynamic systems.6 We present here an enzyme-enabled
dynamic mixture of interconverting cyclodextrins and show
for the rst time the use of a series of templates to selectively
obtain different products from an enzyme-mediated dynamic
Fig. 1 Concept of enzyme-mediated dynamic combinatorial chem-
istry for selective cyclodextrin synthesis. Cyclodextrin glucano-
transferase (CGTase) is used to generate a dynamic mixture of
cyclodextrins starting from various a-1,4-D-glucans. Addition of suit-
able templates induces a change in cyclodextrin distribution to
selectively produce the ‘native’ cyclodextrins (either a-, b-, or g-CD) or
access ‘large-ring’ cyclodextrins formed from 9 and 10 glucose units.
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800
Kongens Lyngby, Denmark. E-mail: sopbee@kemi.dtu.dk
† Electronic supplementary information (ESI) available: Experimental details,
supporting Fig. 1–10. See DOI: 10.1039/c9sc03983j
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In recent decades, chemists have developed more and more amylose, these products build up over a period of time and large-
complex articial dynamic chemical systems, exploring different ring cyclodextrins (CD9 to CD60, with degrees of polymerization of
reversible reactions to enable self-assembly ‘at equilibrium’ and 9 to 60) have been observed briey in the early stages of CGTase
more recently ‘out-of-equilibrium’.7 In principle, reversible action on amylose.13 We hypothesized that since cyclodextrin
enzyme-catalyzed reactions are ideal for use in dynamic systems, formation appears to be dynamic, it might be possible to target
as they work under mild, biologically relevant conditions. specic products from this reaction, including large-ring cyclo-
Enzymes are easily denatured, rendering the library static and the dextrins, using a thermodynamic templating effect.
products isolable. The majority of enzymes, however, have evolved
Herein we present the selective templated synthesis of
to efficiently catalyze unidirectional reactions, making many of cyclodextrins, exploiting CGTase to establish a dynamic chem-
them, at rst glance, unsuitable for use in dynamic chemical ical system of linear and cyclic oligosaccharides (Fig. 1). CGTase
systems. Using optimized conditions, however, dynamic mixtures catalyzes not only reversible transglycosylation but also irre-
of short peptides have been reported, generated using peptidase- versible hydrolysis.13,14 Nevertheless, we show that a kinetically-
catalyzed reversible amide formation6a,c,8 and an aldolase has been trapped yet dynamic mixture of interconverting cyclodextrins
employed to form small DCLs of sialic acid analogues.6b,9
can form, which operates transiently under pseudo-thermody-
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a gly- namic control. By addition of carefully chosen templates, we
cosyltransferase that catalyses the scrambling of a(1 / 4) glyco- can either shi the library composition to produce almost
sidic linkages between the D-glucopyranose monomers of naturally exclusively a-, b-, or g-CD, or we can entirely alter the outcome of
occurring a-1,4-D-glucans (e.g. maltooligosaccharides or amylose). this enzymatic transformation to obtain larger ring cyclodex-
Importantly, CGTase can cyclize linear a-1,4-glucans to form trins, d-CD (CD9) and 3-CD (CD10). The distribution of the
cyclodextrins (CDs), in particular, a-CD, b-CD and g-CD, which are different cyclodextrins generated in the presence of a template
macrocycles formed from 6, 7, and 8 glucose monomers, respec- can be predicted using knowledge of the formation and binding
tively.10 Because of their ability to complex hydrophobic guests, constants, and controlled sequential interconversion of cyclo-
good aqueous solubilities, high stabilities and low toxicities, these dextrins can be achieved in a single reaction vessel.
so-called ‘native’ cyclodextrins have found industrial application
in many elds, and their supramolecular host–guest chemistries
are well-established.11 The industrial production of cyclodextrins is
Results and discussion
frequently performed in the presence of different complexing
pseudo-Thermodynamic control in an out-of-equilibrium
agents, which precipitate either a, b, or g-CD preferentially in
system
order to alter the cyclodextrin product ratio.12 While a-CD, b-CD
The enzyme-promoted reversible formation and interconver-
sion of cyclodextrins was investigated by examining the action
and g-CD are the major cyclic products of the action of CGTase on
Fig. 2 Thermodynamic control over dynamic cyclodextrin systems. (a) Cyclodextrin distribution (left axis, solid lines) and total cyclodextrin
concentration by weight (right axis, dashed lines) as a function of time, determined by HPLC-ELS analysis, when CGTase acts on different a-1,4-
glucans, as indicated (10 mg mLꢀ1), in 50 mM sodium phosphate buffer at pH 7.5. Data points are connected by lines to guide the eye. (b) CGTase
catalyzes both reversible inter- and intramolecular glycosyl transfer of a-1,4-glucans and irreversible hydrolysis of linear and cyclic a-1,4-glucans
(n $ 0; m $ 0; x $ 1; y $ 0; x $ z $ 1). Green hexagons represent a(1 / 4)-linked D-glucopyranose units. The reducing-end (hemiacetal) glucose
units are highlighted in red.
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of CGTase from B. macerans on a-CD, b-CD, g-CD and malto-
hexaose (G6) (Fig. 2a). The reactions were carried out at 25 ꢁC in
sodium phosphate buffer at pH 7.5, which are mild reaction
conditions optimised to favour thermodynamic control over the
system and ensure that the enzyme remained active in solution
for at least four weeks. The reactions where monitored using
high performance liquid chromatography with evaporative
light-scattering detection (HPLC-ELS), which enables detection
of the otherwise chromophore- and uorophore-less oligosac-
charides. The reactions each evolved to produce a mixture of
cyclodextrins (a-, b- and g-CD) and linear a-1,4-glucans (G1–G8
were detectable). Within a matter of 1–7 hours, depending on
the starting material, a steady distribution amongst the three
cyclodextrins was reached. In each case, an equilibrium distri-
bution of ca. 55–57% b-CD, 37–39% a-CD, and 6–7% g-CD was
obtained, which indicated that the production of cyclodextrins
was not only dynamic, but that the cyclodextrin distribution was
determined by their relative stabilities—i.e., that their produc-
tion takes place under thermodynamic control. Essentially, an
enzyme-mediated DCL of cyclodextrins appeared to form.
Unlike in traditional synthetic DCLs, where the covalent
linkages between all building blocks can in principle be formed/
cleaved or exchanged, the pathways for interconversion of
oligomers in enzyme-mediated DCC is complicated by multi-
reactivity and substrate specicity.10c,e,f,13 CGTase catalyzes
both the inter- and intramolecular reversible glycosyl transfer of
a-1,4-glucans, as well as the irreversible hydrolysis of a-1,4-
glucans (Fig. 2b). Furthermore, CGTase can catalyze the trans-
glycosylation of some, but not all, a-1,4-glucans (e.g. glucose can
act as a glycosyl acceptor but not donor, and linear a-1,4-
glucans with less than 7 glucose residues do not undergo
macrocycle-forming intramolecular transglycosylation). Inter-
conversion of cyclodextrins requires a sequence of specic
reactions involving the formation of linear oligosaccharide
intermediates. In our experiments, the total concentration of
cyclodextrins decreased over time (Fig. 2a grey dashed line, ꢂ)
due to a steady rise in the concentration of linear a-1,4-glucans.
The DCL of cyclodextrins exists only transiently (approx. 1 day).
Over a period of several weeks we observed the eventual irre-
versible hydrolysis of all glycosidic linkages (Fig. 3a and ESI
Fig. 1†).
Fig. 3 Cyclodextrins are kinetically trapped products of CGTase action
on a-1,4-glucans, forming a transient DCL in the initial phases of the
reaction. (a) Distribution of both linear (G1–G8) and cyclic (a-, b-, g-CD)
a-1,4-glucans produced over time when CGTase acts on a-CD.
(b) Energy diagram visualizing the suggested energy landscape of the
dynamic system. a-, b-, and g-CD are kinetically trapped in local
minima with surpassable energy barriers for their interconversion, but
G1 is the thermodynamic product and located in the deepest well.
materials (a-CD, b-CD, g-CD and G6) (ESI Fig. 2a†). The
observed lower conversion of G6 to cyclodextrins is then not
a consequence of faster hydrolysis. It is rather related to the fact
that 1/6th of the glucose residues in G6 (namely the reducing-
end glucose) cannot be utilized to form cyclodextrins (so the
highest possible theoretical yield of cyclodextrins from G6
would be 5/6 z 83%). Furthermore, for G6, hydrolysis imme-
diately generates very short maltooligosaccharides, from which
there is a low probability that intermolecular glycosyl transfer
will lead to linear a-1,4-glucans long enough to then cyclize and
form cyclodextrins.
We thus reached the conclusion that although glucose is the
true thermodynamic product of the action of CGTase on a-1,4-
glucans, a-CD, b-CD and g-CD must exist within a kinetically
trapped sub-system over which there is pseudo-thermodynamic
control. Fig. 3b suggests a picture of the overall energy land-
scape in the system. The energy barrier for the interconversion
of cyclodextrins is readily overcome, so that thermodynamic
control exists transiently over this sub-system. As with all
kinetically trapped systems, the starting point from which
equilibrium is approached (i.e. the particular a-1,4-glucan
supplied to the system) affects the degree to which the system
becomes kinetically trapped (i.e. the overall cyclodextrin
The evolution of the dynamic systems, and the total
concentration of cyclodextrins present, when a steady distri-
bution was achieved, varied with the starting material. The
reaction with G6 produced signicantly lower concentrations of
cyclodextrins (Fig. 2a and ESI Fig. 1d†). We therefore chose to
investigate the rate of irreversible hydrolysis when CGTase acts
upon a-CD, b-CD, g-CD and G6 by monitoring the build-up of
reducing-end (hemiacetal) glucose residues. While hydrolysis
generates a range of linear products that are fed back into the
dynamic system to produce cyclodextrins, each hydrolytic
cleavage results in a new reducing-end glucose, which cannot be
reincorporated into a cyclodextrin (Fig. 2b).15 The hydrolysis
reaction was found to follow second order reaction kinetics with
respect to the decreasing concentration of hydrolysable glyco-
sidic linkages, and almost identical rate constants (k z 1.1 ꢂ
10ꢀ3 Mꢀ1 sꢀ1) were measured for the four different starting
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concentration and the lifetime of the transient DCL). So long as CD9–CD11 can bind dodecaborate cluster ions with high
thermodynamic control exists transiently, however, there is the affinity and some selectivity.19 We therefore treated a-CD with
opportunity to employ templates to stabilize and amplify CGTase in the presence of Cs2B12I12 (4) and observed
specic oligomeric products within the system.
a remarkable shi in product distribution (Fig. 4e and ESI
Fig. 6†). At pseudo-equilibrium, two new peaks were observed in
the HPLC-ELS chromatogram and identied as CD9 and CD10
(by MALDI-TOF-MS analysis following fractional collection of
the HPLC-separated compounds). These new peaks from the
larger cyclodextrins CD9 and CD10 made up 13% and 4% of the
total area of all cyclodextrin peaks, respectively, aer 6 hours of
reaction, whereas they were practically unobservable in the
absence of the template. A similarly high yield of CD10 has
recently been reported, but using a genetically engineered
CGTase.20 CD9 has been isolated in only 0.04% yield using
a native CGTase.18a We found that despite the higher reported
Templated selective cyclodextrin synthesis
By utilizing templates known to bind strongly and selectively to
cyclodextrins in aqueous solution we could successfully
promote the synthesis of a-, b- or g-CD with high selectivity. We
found that when a-CD (10 mg mLꢀ1) was treated with CGTase in
the presence of sodium dodecyl sulfate (SDS) (1) (10 mM),15 a-
CD made up 83% of the total cyclodextrin concentration at
pseudo-equilibrium (Fig. 4b), compared with 38% in the
absence of template (Fig. 4a). The use of 1-adamantane
carboxylic acid (2)16 as a template resulted in the formation of
almost exclusively b-CD (>99% of total CDs, Fig. 4c). Templating
with NaBPh4 (3)17 selectively yielded g-CD (>99% of total CDs,
Fig. 4d). Identical distributions of cyclodextrins in the presence
of templates were obtained irrespective of which a-1,4-glucan
was used as the starting material (a-, b-, g-CD or G6) (ESI Fig. 3–
5†), which is further evidence of pseudo-thermodynamic
control.
affinities of CD9 (Ka ¼ 6.8 ꢂ 105 Mꢀ1) and CD10 (Ka ¼ 2.1 ꢂ 106
2ꢀ
Mꢀ1) for the B12 12
I
dianion compared with g-CD (Ka ¼ 6.7 ꢂ
104 Mꢀ1),19,21 g-CD was the major cyclodextrin product gener-
ated in this reaction. Product distribution in a DCL is deter-
mined not only by the relative affinities of the library members
for a given template, but also the intrinsic stability of the
different library members.22 That CD9 and CD10 are not
observed in the absence of a template is indicative of their much
smaller formation constants. Their templated synthesis is
therefore signicantly more challenging and requires particu-
larly strong interactions with a template. At such high affinities
it also becomes increasingly likely that library members can
The supramolecular chemistry of large-ring cyclodextrins is
relatively unexplored. They are accessible only aer extensive
chromatographic separation from complex mixtures containing
numerous cyclodextrins of different sizes formed when amylose
is briey exposed to CGTase.13,18 It was recently reported that
Fig. 4 Template-directed enzyme-mediated dynamic combinatorial synthesis of cyclodextrins. (a–e) Chromatograms (HPLC-ELS) showing the
pseudo-equilibrium distribution of cyclodextrins produced when CGTase acts on a-CD in the absence or presence of templates 1–4 (10 mM). (f)
Model employed to simulate a DCL of cyclodextrins and predict the concentrations of a-, b-, and g-CD in the presence of SDS (1). (g) Comparison
between the predicted and experimentally determined cyclodextrin distributions generated in the presence of different concentrations of SDS (1).
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become kinetically trapped in the presence of the template. concentrations of SDS (1) (Fig. 4g). An improved 89% selectivity
Although the highly selective synthesis achieved for a-, b- and g- was obtained when the template concentration was lowered
CD could not be replicated for CD9 and CD10, this result from 10 mM to 5 mM.
demonstrates the possibility to obtain entirely new products
To understand this result, the binding interaction between
from enzymatic reactions via a thermodynamic template effect. SDS (1) and each cyclodextrin was investigated individually by
1
It is noteworthy that in the presence of templates, higher means of H NMR spectroscopy titrations (ESI Fig. 8–10†). The
overall yields of cyclodextrins were observed and the build-up of resulting binding isotherms clearly showed that SDS (1) can
linear products was slower for all starting materials. The rate of bind two molecules of both a-CD and b-CD. For a-CD, two
CGTase-catalyzed irreversible hydrolysis of the different a-1,4- strong binding interactions were observed (Ka1 ¼ (1.9 ꢃ 0.5) ꢂ
glucans in the presence of 1-adamantane carboxylic acid was 104 Mꢀ1 and Ka2 ¼ (2.3 ꢃ 0.4) ꢂ 104 Mꢀ1), while for b-CD
thus investigated. Signicantly smaller apparent second order a comparably high rst binding constant was determined but
rate constants (ca. 45-fold lower) were determined (ESI Fig. 2b†), the second binding event was markedly weaker (Ka1 ¼ (1.6 ꢃ
compared with those for hydrolysis in the absence of template. 0.4) ꢂ 104 Mꢀ1 and Ka2 ¼ (7 ꢃ 4) ꢂ 102 Mꢀ1). Using the obtained
It appears that the cyclodextrins are ‘protected’ against hydro- binding constants, along with relative formation constants for
lysis by host–guest complex formation.
a-, b-, and g-CD, determined from the pseudo-equilibrium
distribution of these cyclodextrins in the absence of template,
we simulated a DCL of cyclodextrins according to the model
shown in Fig. 4f using the DCLsim soware previously devel-
oped in the Otto group.22a The expected distribution of cyclo-
dextrins in the presence of varying concentrations of SDS were
calculated and it was predicted that the highest yield of a-CD
would in fact be obtained at a lower template concentration, in
line with our experimental results (Fig. 4g). We could rationalize
this result on the basis of a 2 : 1 binding mode, as a 5 mM
template concentration equates to approximately one molecule
of SDS (1) per two molecules of a-CD. That the DCLsim soware,
developed to predict the outcome of a network of chemical
equilibria operating under thermodynamic control, could
successfully predict the distribution of cyclodextrins in our
system supports the experimental evidence that pseudo-
DCL simulation
Unsatised with the obtained 83% selectivity for a-CD achieved
using SDS (1) as a template, we sought to improve this result.
Screening of a series of different amphiphiles revealed that
templates with longer aliphatic chains were more effective at
amplifying a-CD, but b-CD was still consistently obtained as
a side product (ESI Fig. 7†). We suspected that the inability to
completely switch the cyclodextrin distribution to a-CD was not
because a-CD bound too weakly to 1, but rather due to
a competing interaction between 1 and b-CD. As it has been
shown that in DCLs where several oligomers can bind the
template, the distribution of the different oligomers formed is
inuenced by the concentration of the template,22 we investi-
gated the action of CGTase on a-CD in the presence of varying
Fig. 5 Sequential interconversion of cyclodextrins. Cyclodextrin distribution (left axis, solid lines) and total cyclodextrin concentration (right axis,
dashed line) as a function of time when different templates (NaBPh4 (3), SDS (1) and 1-adamantane carboxylic acid (2)) were added sequentially,
and in one pot, to a dynamic cyclodextrin system generated by the action of CGTase on maltooctaose (G8). Data points are connected by lines to
guide the eye. Conditions and analysis as in Fig. 2.
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thermodynamic control exists transiently over this subsystem of that interconvert products and substrates via a series of reaction
kinetically trapped cyclodextrins.
steps. Employing templates to modulate the ne balance
between kinetic and thermodynamic control in enzyme-
mediated systems offers a new approach for synthetic (bio)
chemists to control enzyme reactivity and selectivity, and obtain
new, or difficult-to-access, structures and materials.
Sequential cyclodextrin interconversion
To further showcase the extent to which we can use templates
to control the outcome of this dynamic cyclodextrin system, we
sought to switch sequentially between the three ‘native’ cyclo-
dextrins in a single reaction vessel (Fig. 5). Maltooctaose (G8)
was exposed to CGTase (under conditions identical to previous
experiments) and allowed to react and reach a pseudo-equilib-
rium cyclodextrin mixture before addition of one equivalent of
NaBPh4 (3). Within six hours, the cyclodextrin composition
switched to almost exclusively g-CD (ca. 95%). At this time, SDS
(1) was added in large excess, which, as anticipated, caused
a change in the cyclodextrin composition to produce primarily
a-CD (ca. 60%) within 20 hours. Due to the lower affinity and
selectivity of 1, compared with the other templates, it was not
possible to push the cyclodextrin distribution to a higher a-CD
concentration. 1-Adamantane carboxylic acid (2) was then
added in excess, and within 20 hours the cyclodextrin compo-
sition had switched to nearly exclusively b-CD (ca. 95%).
Remarkably, CGTase remained active over several days and in
the presence of high concentrations of the various templates.
Furthermore, cyclodextrins accounted for ca. 70% of the glucan
material aer 54 hours, compared with ca. 50% aer only 8
hours in the untemplated reaction starting from G6 (Fig. 2a). As
cyclodextrins are presumably not substrates for the enzyme
when bound to the templates, the presence of a template
reduces the concentration of available substrate in solution.
This leads to a longer equilibration time for the interconver-
sion of cyclodextrins (note the time-axis in Fig. 5), but also
a reduced rate of hydrolysis, leading to a longer-lasting tran-
sient DCL of cyclodextrins operating under pseudo-thermody-
namic control.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We are grateful for the kind gi of the CGTase enzyme isolated
from Bacillus macerans provided by Amano Enzyme Inc.,
Nagoya, Japan. We gratefully acknowledge the Villum Founda-
tion, Carlsberg Foundation and Technical University of Den-
mark for nancial support, and Associate Professor M.
Pittelkow, University of Copenhagen, for use of his group's ESI-
MS system.
Notes and references
1 S. Otto, R. L. E. Furlan and J. K. M. Sanders, Science, 2002,
297, 590–593.
2 K. S. Chichak, S. J. Cantrill, A. R. Pease, S.-H. Chiu,
G. W. V. Cave, J. L. Atwood and J. F. Stoddart, Science,
2004, 304, 1308–1312.
3 S. Zarra, D. M. Wood, D. A. Roberts and J. R. Nitschke, Chem.
Soc. Rev., 2015, 44, 419–432.
4 N. Ponnuswamy, F. B. L. Cougnon, J. M. Clough, G. D. Panto¸s
and J. K. M. Sanders, Science, 2012, 338, 783–785.
5 (a) J.-M. Lehn, Chem. Soc. Rev., 2017, 36, 151–160; (b) J. Li,
P. Nowak and S. Otto, J. Am. Chem. Soc., 2013, 135, 9222–
9239.
6 (a) P. G. Swann, R. A. Casanova, A. Desai, M. M. Frauenhof,
M. Urbancic, U. Slomczynska, A. J. Hopnger, G. C. Le
Breton and D. L. Venton, Pept. Sci., 1996, 40, 617–625; (b)
R. J. Lins, S. L. Flitsch, N. J. Turner, E. Irving and
S. A. Brown, Angew. Chem., Int. Ed., 2002, 41, 3405–3407; (c)
R. J. Williams, A. M. Smith, R. Collins, N. Hodson,
A. K. Das and R. V. Ulijn, Nat. Nanotechnol., 2009, 4, 19–24.
7 E. Mattia and S. Otto, Nat. Nanotechnol., 2015, 10, 111–119.
8 C. G. Pappas, R. Sha, I. R. Sasseli, H. Siccardi, T. Wang,
V. Narang, R. Abzalimov, N. Wijerathne and R. V. Ulijn,
Nat. Nanotechnol., 2016, 11, 960–967.
Conclusions
In summary, we have exploited CGTase to generate dynamic
mixtures of interconverting cyclodextrins operating under
pseudo-thermodynamic control, from which we could template
the selective synthesis of a-CD, b-CD or g-CD, and access
otherwise inaccessible large-ring cyclodextrins. This work
shows for the rst time the systematic use of articial templates
to selectively access different target products from a system of
enzyme-catalyzed reactions, and thus demonstrates unprece-
dented control over, and the ability to redirect the outcome of
enzymatic reactions.
9 R. J. Lins, S. L. Flitsch, N. J. Turner, E. Irving and S. A. Brown,
Tetrahedron, 2004, 60, 771–780.
We believe that combining enzyme-mediated reactions with 10 (a) F. Schardinger, Z. Unters. Nahr.- Genussm.
thermodynamic templating represents an important step
forward in directed self-assembly. Enzymes offer new opportu-
nities as they can generate dynamic systems from seemingly
inert molecules. The approach is not limited to carbohydrate-
modifying enzymes. We foresee that a range of enzymes could
be employed for templated enzymatic synthesis and also that
enzyme-mediated dynamic systems could be generated using
sets of complementary enzymes catalysing irreversible reactions
Gebrauchsgegenstaende, 1903, 6, 865–880; (b) D. French and
R. E. Rundle, J. Am. Chem. Soc., 1942, 64, 1651–1653; (c)
D. French, J. H. Pazur, M. L. Levine and E. Norberg, J. Am.
Chem. Soc., 1948, 70, 3145; (d) D. French, D. Knapp and
J. H. Pazur, J. Am. Chem. Soc., 1950, 72, 5150–5152; (e)
J. C. M. Uitdehaag, B. A. van der Veen, L. Dijkhuizen and
B. W. Dijkstra, Enzyme Microb. Technol., 2002, 30, 295–304;
(f) J. C. Uitdehaag, K. H. Kalk, B. A. van der Veen,
9986 | Chem. Sci., 2019, 10, 9981–9987
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
L. Dijkhuizen and B. W. Dijkstra, J. Biol. Chem., 1999, 274,
34868–34876; (g) G. Crini, Chem. Rev., 2014, 114, 10940–
10975.
11 M. V. Rekharsky and Y. Inoue, Chem. Rev., 1998, 98, 1875–
1918.
12 (a) A. Biwer, G. Antranikian and E. Heinzle, Appl. Microbiol.
Biotechnol., 2002, 59, 609–617; (b) Z. Li, M. Wang, F. Wang,
Z. Gu, G. Du, J. Wu and J. Chen, Appl. Microbiol.
Biotechnol., 2007, 77, 245–255.
13 Y. Terada, M. Yanase, H. Takata, T. Takaha and S. Okada, J.
Biol. Chem., 1997, 272, 15729–15733.
Chemical Science
´
K. Gessler, I. Uson, T. Takaha, N. Krauss, S. M. Smith,
S. Okada, G. M. Sheldrick and W. Saenger, Proc. Natl. Acad.
Sci. U. S. A., 1999, 96, 4246–4251; (c) J. Jacob, K. Geßler,
D. Hoffmann, H. Sanbe, K. Koizumi, S. M. Smith,
T. Takaha and W. Saenger, Angew. Chem., Int. Ed., 1998,
37, 605–609; (d) I. Miyazawa, H. Ueda, H. Nagase, T. Endo,
S. Kobayashi and T. Nagai, Eur. J. Pharm. Sci., 1995, 3, 153–
162.
19 K. I. Assaf, D. Gabel, W. Zimmermann and W. M. Nau, Org.
Biomol. Chem., 2016, 14, 7702–7706.
20 C. Sonnendecker, S. Thurmann, C. Przybylski,
¨
14 Y. B. Tewari, R. N. Goldberg and M. Sato, Carbohydr. Res.,
1997, 301, 11–22.
15 S. Kobayashi, K. Kainuma and D. French, J. Jpn. Soc. Starch
Sci., 1983, 30, 62–68.
16 W. C. Cromwell, K. Bystrom and M. R. Eink, J. Phys. Chem.,
1985, 89, 326–332.
F. D. Zitzmann, N. Heinke, Y. Krauke, K. Monks,
A. A. Robitzki, D. Belder and W. Zimmermann, Angew.
Chem., Int. Ed., 2019, 58, 6411–6414.
21 K. I. Assaf, M. S. Ural, F. Pan, T. Georgiev, S. Simova,
K. Rissanen, D. Gabel and W. M. Nau, Angew. Chem., Int.
Ed., 2015, 54, 6852–6856.
17 T. Nhujak and D. M. Goodall, Electrophoresis, 2001, 22, 117– 22 (a) P. T. Corbett, S. Otto and J. K. M. Sanders, Chem.–Eur. J.,
122.
2004, 10, 3139–3143; (b) P. T. Corbett, J. K. M. Sanders and
S. Otto, J. Am. Chem. Soc., 2005, 127, 9390–9392.
18 (a) K. Koizumi, H. Sanbe, Y. Kubota, Y. Terada and
T. Takaha, J. Chromatogr. A, 1999, 852, 407–416; (b)
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