Enzymatic synthesis and photoswitchable enzymatic cleavage of a peptide-linked rotaxane

Article abstract of DOI:10.1002/anie.200504064

(Figure Presented) Selective digestion: α-Chymotrypsin was used to synthesize a cyclodextrin azo dye rotaxane and to digest it back to its components. E → Z photoisomerization of the rotaxane completely changes its affinity for the enzyme. The presence of

Full text of DOI:10.1002/anie.200504064

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Rotaxanes
ester acts as an acyl donor and gives an acyl enzyme
intermediate, which then acylates the amine to form the
peptide bond. A variety of serine and cysteine proteases can
be used, and we chose the serine protease a-chymotrypsin
because it is readily available and requires no preactivation.
To effect efficient peptide coupling, the acyl donor (ester) and
acyl acceptor (amine) must be tailored to suit the active site of
the enzyme and to match the S1 and S1’ specificities (S1 and S1’
are the binding sites for amino acid residues on the donor and
acceptor sides of the new amide bond, respectively).[7] For a-
chymotrypsin, the S1 site prefers large hydrophobic side
chains, while the S1’ site prefers cationic amino acid residues.[8]
Accordingly, a phenylalanine-based donor, MalPheOMe, and
an arginine-based acceptor, (E)-2, were chosen as the
substrates for our enzyme-catalyzed rotaxane synthesis. The
arginine-substituted azo dye, (E)-2, was synthesized in four
steps from the phenol 3, as shown in Scheme 1.
DOI: 10.1002/anie.200504064
Enzymatic Synthesis and Photoswitchable
Enzymatic Cleavage of a Peptide-Linked
Rotaxane**
Andrew G. Cheetham, Michael G. Hutchings,
Tim D. W. Claridge, and Harry L. Anderson*
Cyclodextrin inclusion complexes often exhibit useful proper-
ties, such as enhanced chemical stability, fluorescence effi-
ciency, and solubility.[1] An inclusion complex of this type can
be converted into a kinetically robust rotaxane[2] by attaching
bulky stoppers to both ends of the threaded guest, perma-
nently locking it inside the cyclodextrin.[3] The hydrophobic
effect is the main driving force for threading, so it is
convenient to synthesize these rotaxanes in water, although
this limits the range of chemistry available for covalent
coupling. Enzymes are uniquely suited for catalyzing cova-
lent-bond formation in water, so it seems surprising that they
have not previously been used to catalyze rotaxane synthesis.
Herein we report the chymotrypsin-catalyzed synthesis of a
peptide-linked azo dye rotaxane, (E)-1ꢀa-CD (CD = cyclo-
dextrin). Rotaxanes of this type generally exhibit reversible
photochemical cis-trans isomerization, with concomitant
relocation of the cyclodextrin along the axle of the rotaxane.[4]
Herein we show that photoisomerization of (E)-1ꢀa-CD can
be used to prevent its chymotrypsin-catalyzed hydrolysis.
Although there are no previous examples of enzyme-cata-
lyzed rotaxane synthesis, enzyme-catalyzed rotaxane cleavage
has been studied in detail as an approach to drug delivery in
which the enzymes naturally present are used to liberate the
components of the rotaxane.[5] Photoswitchable rotaxane
cleavage is relevant to this area, because it could enable a
drug to be delivered selectively to cells that have a specific
enzyme and that are being irradiated with light of the
appropriate wavelength.
Scheme 1. Enzymatic synthesis and hydrolysis of the rotaxane (E)-
1ꢀa-CD. Boc-Arg(Pbf)-OH = Na-tert-butoxycarbonyl-Nw-(2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl)-l-arginine; DMF = N,N-
dimethylformamide; HEPES = 4-(2-hydroxyethyl)piperazine-1-ethane-
sulfonic acid; Mal = maleyl; pybop = (benzotriazol-1-yloxy)tripyrroli-
dinophosphonium hexafluorophosphate; TFA = trifluoroacetic acid.
Protease-catalyzed synthesis of peptides has been inves-
tigated extensively because it offers distinct advantages over
conventional chemical synthesis, such as high selectivity and
lack of racemization.[6] The most common approach is
amination of an amino acid ester under kinetic control. The
The a-chymotrypsin-catalyzed acylation of (E)-2 was
monitored by HPLC (Figure 1). Use of excess acyl donor
(10 equiv) and excess a-cyclodextrin (15 equiv) gave 87%
conversion into the rotaxane (E)-1ꢀa-CD after 20 min.
However, at longer reaction times the enzyme hydrolyzes
the rotaxane back to (E)-2 by selective cleavage of the
PheArg link. Stopping the reaction after 20 min and purifi-
cation by reversed-phase and anion-exchange chromatogra-
phy enabled the rotaxane to be isolated in 37% yield. The
same reaction in the absence of cyclodextrin gave the
dumbbell peptide azo dye (E)-1 in 48% yield. When a
dumbbell with non-equivalent ends, such as (E)-1, is threaded
through a cyclodextrin with non-equivalent rims, there is the
[*] A. G. Cheetham, Dr. T. D. W. Claridge, Prof. H. L. Anderson
Department of Chemistry, Chemistry Research Laboratory
University of Oxford
12 Mansfield Road, Oxford, OX1 3TA (UK)
Fax: (+44)1865-28-5002
E-mail: harry.anderson@chem.ox.ac.uk
Dr. M. G. Hutchings
DyStar UK Ltd., School of Chemistry
University of Manchester
Oxford Road, Manchester, M13 9PL (UK)
[**] We thank the Engineering and Physical Sciences Council (EPSRC)
and DyStar UK Ltd. for supporting this work.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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from the azo region to the phenoxyethyl moiety adjacent to
the dipeptide stopper. For example, in (Z)-1ꢀa-CD, HC shows
no NOE interactions with cyclodextrin protons, whereas HF
and HH show NOE interactions with 3-H and 5-H, respec-
tively (Figure 2a).
Figure 1. HPLC data showing the formation of rotaxane (E)-1ꢀa-CD
over time, as in Scheme 1. All concentrations are normalized by
dividing by the initial concentration of (E)-2. The conversion into
(E)-1ꢀa-CD peaks at 87% after 20 min, whereas the concentration of
(E)-1 peaks at 16% after 150 min, and the concentration of (E)-2
reaches a minimum of 2.5% after 20 min. The concentration of
MalPheOMe (not plotted here)becomes less than 1% of its initial
value after 20 min. Initial concentrations: [(E)-2]=1.57 mm, [MalPheO-
Me]=15.7 mm, [a-CD]=23.6 mm, [a-chymotrypsin]=0.3 mm. Buffer:
HEPES 0.1m, NaCl 0.2m, CaCl2 0.02m, pH 8.
possibility of forming two stereoisomers. HPLC analysis
indicates that rotaxane (E)-1ꢀa-CD is formed as a single
isomer, and this observation was supported by 1H and
13C NMR spectroscopic analysis. 1H NMR NOESY experi-
ments showed that the wide 2,3-rim of the cyclodextrin is
directed towards the sulfonate groups, while the narrower 6-
rim is directed towards the peptide. For example, HC shows a
strong NOE interaction with 3-H of the cyclodextrin (and a
weak one with 5-H, but none with 6-H or 6’-H), whereas HF
shows strong NOE interactions with 5-H, 6-H and 6’-H, but
none with 3-H. Surprisingly, NOE investigations of the (E)-
2ꢀa-CD inclusion complex revealed that the dominant
isomer of this complex has the opposite cyclodextrin orienta-
tion,[9] implying that the enzyme determines the stereochem-
istry of the rotaxane by kinetic selection of the less stable
isomer of this inclusion complex.
Figure 2. a)Co-conformation of ( Z)-1ꢀa-CD deduced from NOE
measurements. b)Schematic representation of photoisomerization
and enzyme-catalyzed cleavage of the PheArg peptide bond, which
decreases the bulk of the end group and results in unthreading of the
cyclodextrin (l1 =368 nm, l2 =445 nm).
The relative rates of enzyme-catalyzed hydrolysis of (E)-
1ꢀa-CD, (Z)-1ꢀa-CD, (E)-1, and (Z)-1 were tested by
monitoring the hydrolysis of a mixture of these four
compounds simultaneously by HPLC, to test whether photo-
switching can be used to control enzymatic hydrolysis
(Figure 2b). The reaction was carried out at 277 K in the
dark at high enzyme concentration to minimize thermal or
photochemical Z!E isomerization. The data from this
experiment were fitted to first order decay curves to give
the pseudo-first-order rate constants indicated in Figure 3.[10]
The E and Z isomers of the dumbbell 1 are hydrolyzed at the
same rate, whereas (E)-1ꢀa-CD is hydrolyzed more than 80
times faster than (Z)-1ꢀa-CD. The slower hydrolysis of the
Z rotaxane shows that when the cyclodextrin shifts towards
the peptide link it hinders interaction with the enzyme; thus
the reactivity of the peptide can be controlled by photo-
isomerization. (Z)-1ꢀa-CD is hydrolyzed so slowly that,
under the conditions of this experiment, thermal Z!E
isomerization dominates, and we can only estimate an upper
limit to its rate of hydrolysis.
The E!Z photoisomerization of (E)-1ꢀa-CD and (E)-1
1
in aqueous solution was monitored by HPLC and H NMR
spectroscopy. Irradiation at 368 nm gave photostationary
mixtures dominated by the Z isomers, whereas irradiation at
445 nm rapidly shifted the composition back towards the
E isomers. This photochemistry is cleanly reversible and no
by-products were detected by HPLC or NMR spectroscopy.
The rate of dark thermal Z!E isomerization was measured
for both the rotaxane (Z)-1ꢀa-CD and the dumbbell (Z)-1 in
aqueous solution at 297 K and 277 K. In each case, the
reaction is first order, and isomerization is faster in the
rotaxane (t1/2 = 5.4 ꢁ 0.3h at 297 K and 72 ꢁ 10 h at 277 K)
than in the dumbbell (t1/2 = 34 ꢁ 3h at 297 K and > 200 h at
277 K). Thus the presence of the cyclodextrin destabilizes the
Z isomer of the azo dye and decreases the activation energy
for thermal isomerization (as noticed in similar rotaxanes by
Nakashima and co-workers[4b]). NOE analysis of (E)-1ꢀa-CD
and (Z)-1ꢀa-CD in D2O showed that E!Z photoisomeriza-
tion of the rotaxane results in relocation of the a-cyclodextrin
It seems amazing that the bulky rotaxane (E)-1ꢀa-CD is
hydrolyzed 2.6 times faster than the corresponding dumbbell
(E)-1 (Figure 3). This implies that the cyclodextrin increases
the affinity of the peptide for the active site of chymotryp-
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Communications
cyclodextrin.[12] Interactions of this type may explain why the
presence of the cyclodextrin increases the affinity of the
peptide for the enzyme.
In conclusion, we have shown that enzymes can be used to
synthesize cyclodextrin rotaxanes rapidly and selectively
under mild conditions. This chemistry should be useful for
the synthesis of encapsulated dyes[3] and insulated molecular
wires.[13] The observation that photoisomerization of (E)-
1ꢀa-CD turns off its reactivity towards enzyme-catalyzed
hydrolysis can also be regarded as a first step towards
photoswitchable rotaxane-mediated drug delivery.
Received: November 15, 2005
Published online: February 3, 2006
Figure 3. Plot of normalized concentration (c/c0)of ( E)-1ꢀa-CD (&),
~
&
*
(Z)-1ꢀa-CD ( ) , (E)-1 ( ), and (Z)-1 ( )against time ( t)during
competitive a-chymotrypsin-catalyzed hydrolysis, showing values of
pseudo-first-order rate constants. The reaction was monitored by
HPLC at 277 K in the dark; initial concentrations: [1ꢀa-CD]=1.2 mm,
[1]=1.2 mm and [a-chymotrypsin]=12 mm.[10] These rate constants
have error margins of about 30%, but their relative values are accurate
to ꢁ5% owing to the nature of the competition experiment.
Keywords: azo compounds · enzyme catalysis · peptides ·
.
photochemistry · rotaxanes
[1] Comprehensive Supramolecular Chemistry, Vol. 3 (Eds: J. L.
Atwood, J. E. D. Davies, D. D. MacNichol, F. Vögtle), Perga-
mon, Oxford, UK, 1996.
sin.[11] This conclusion is supported by the selective formation
of this stereoisomer of the rotaxane (E)-1ꢀa-CD, despite the
fact that the other stereoisomer of the inclusion complex (E)-
2ꢀa-CD predominates in solution. We carried out molecular-
mechanics calculations on the a-chymotrypsin/(E)-1ꢀa-CD
complex (with the PheArg peptide bound to the active site of
the enzyme) to gain insight into the factors stabilizing this
complex. The energy-minimized structure (Figure 4) shows
that the cyclodextrin lies close to the surface of the enzyme
and indicates a hydrogen-bonding interaction between the
OH group of tyrosine 146 and O6 on the narrow rim of the
[2] S. A. Nepogodiev, J. F. Stoddart, Chem. Rev. 1998, 98, 1959 –
1976; F. M. Raymo, J. F. Stoddart, Chem. Rev. 1999, 99, 1643–
1663; A. Harada, Acc. Chem. Res. 2001, 34, 456 – 464.
[3] a) J. E. H. Buston, J. R. Young, H. L. Anderson, Chem.
Commun. 2000, 905 – 906; b) C. A. Stanier, M. J. OꢀConnell, W.
Clegg, H. L. Anderson, Chem. Commun. 2001, 493– 494, 787;
c) M. R. Craig, M. G. Hutchings, T. D. W. Claridge, H. L. Ander-
son, Angew. Chem. 2001, 113, 1105 – 1108; Angew. Chem. Int.
Ed. 2001, 40, 1071 – 1074.
[4] a) H. Murakami, A. Kawabuchi, K. Kotoo, M. Kunitake, N.
Nakashima, J. Am. Chem. Soc. 1997, 119, 7605 – 7606; b) H.
Murakami, A. Kawabuchi, R. Matsumoto, T. Ido, N. Nakashima,
J. Am. Chem. Soc. 2005, 127, 15891 – 15899; c) D.-H. Qu, Q.-C.
Wang, J. Ren, H. Tian, Org. Lett. 2004, 6, 2085 – 2088.
[5] a) M. Eguchi, T. Ooya, N. Yui, J. Controlled Release 2004, 96,
301 – 307; b) T. Ooya, K. Arizono, N. Yui, Polym. Adv. Technol.
2000, 11, 642 – 651; c) T. Ooya, N. Yui, J. Controlled Release 1999,
58, 251 – 269.
[6] a) F. Bordusa, Chem. Rev. 2002, 102, 4817 – 4867; b) V. Schel-
lenberger, H.-D. Jakubke, Angew. Chem. 1991, 103, 1440;
Angew. Chem. Int. Ed. Engl. 1991, 30, 1437 – 1449.
[7] This nomenclature was introduced by I. Schechter, A. Berger,
Biochem. Biophys. Res. Commun. 1969, 37, 157 – 162.
[8] V. Schellenberger, U. Schellenberger, Y. V. Mitin, H.-D.
Jakubke, Eur. J. Biochem. 1990, 187, 163– 167.
[9] UV/Vis titration of (E)-2 and a-cyclodextrin, carried out under
the same conditions as the enzymatic synthesis (HEPES buffer,
pH 8, 228C), gave data which fit well to a 1:1 binding isotherm
with a binding constant of K = 2.5 ꢁ 0.2 ꢁ 103 mꢂ1
.
1H NMR
titration showed the formation of two 1:1 complexes, in a ratio
of 1:10. These complexes undergo slow exchange on the
chemical shift timescale, with each other and with excess (E)-
2, but exchange cross-peaks were observed during NOESY
experiments, and the complexes appeared to form immediately
on addition of the cyclodextrin; hence the exchange rate is
probably in the 1-sꢂ1 regime. The dominant isomer of the (E)-
2ꢀa-CD inclusion complex gave NOE interactions of HC and HD
with both 5-H and 6-H, HF with both 3-H and 5-H, and HG with
3-H, indicating that it has the opposite cyclodextrin orientation
to the (E)-1ꢀa-CD rotaxane. The 1H NMR spectrum of the
minor isomer of the (E)-2ꢀa-CD inclusion complex is very
similar to that of the (E)-1ꢀa-CD rotaxane, leading us to
conclude that these two species have the same cyclodextrin
Figure 4. Calculated structure of the a-chymotrypsin/(E)-1ꢀa-CD
complex, with PheArg bound in the active site of the enzyme. The
tyrosine-146 residue (shown in red, top left of rotaxane)is hydrogen
bonded to O6 of the cyclodextrin. (Other protein residues not
displayed; enzyme surface: 1.4-ꢀ probe radius; a-cyclodextrin
green.)[12]
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Chemie
orientation, and this is supported by the observation of NOE
interactions of HC with 3-H, and HG with 6-H, 6’-H, and 5-H.
[10] The pseudo-first-order rate constants marked in Figure 3are
simply the gradients of the straight-line fits. Under these
conditions the rate constants for thermal Z!E isomerization
of (Z)-1 and (E)-1ꢀa-CD are < 1 ꢁ 10ꢂ6 sꢂ1 and (2.7 ꢁ 0.4) ꢁ
10ꢂ6 sꢂ1, respectively; hence, thermal isomerization makes no
significant contribution to the loss of (Z)-1, but it accounts for at
least 90% of the loss of (Z)-1ꢀa-CD.
[11] The presence of the cyclodextrin in (E)-1ꢀa-CD may accelerate
enzymatic hydrolysis by lowering the Michaelis constant KM or
by increasing the turnover number kcat. We plan to carry out
Michaelis–Menten analysis to address this issue.
[12] The a-chymotrypsin/(E)-1ꢀa-CD complex was modelled by
using the Maestro software (from Schrödinger LLC) starting
from the crystallographic coordinates of a-chymotrypsin (PDB
ID: 6CHA; A. Tulinsky, R. A. Blevins, J. Biol. Chem. 1987, 262,
7737 – 7743) and of the methyl orange a-CD complex (K.
Harata, Bull. Chem. Soc. Jpn. 1976, 49, 1493– 1501). The
structure was minimized by using MacroModel (Schrödinger
LLC), with the MMFFs force field, with water as the solvent, and
with the Polak–Ribier Convergence Gradient Method at a
convergence threshold of 0.005. During this calculation the
geometry of the azo group was constrained to be planar, and the
carbonyl of the Phe residue of the rotaxane was constrained to
be within hydrogen-bonding distance of Ser-195 and Gly-193.
The calculated structure (Figure 4) features the following
=
intercomponent hydrogen bonds: Gly193-NH to C O of Argrotax
=
(2.2 ꢂ); Arg-NH2(1)rotax to C O of Phe-41 (2.0 ꢂ); Arg-NH-
=
=
(Guan.)rotax to C O of Phe-41 (1.8 ꢂ); Arg-NH2(2)rotax to C O of
Cys-58 (1.9 ꢂ); Phe-NHrotax to N-imidazole of His-57 (2.8 ꢂ);
Phe-NHrotax to OH of Ser-195 (2.7 ꢂ); Arg-NHrotax to OH of Ser-
195 (1.7 ꢂ); a-CD 2-OH to 3-SO3 (1.8 ꢂ); Tyr-146-OH to a-CD
6-OH (1.7 ꢂ). Molecular-mechanics calculations were also
performed on both isomers of the (E)-2ꢀa-CD inclusion
complex bound to the enzyme. Similar interactions were
predicted for both isomers; however, the level of these
calculations is not sufficient to probe the origins of the exclusive
formation of one isomer of the rotaxane.
[13] F. Cacialli, J. S. Wilson, J. J. Michels, C. Daniel, C. Silva, R. H.
Friend, N. Severin, P. Samori, J. P. Rabe, M. J. OꢀConnell, P. N.
Taylor, H. L. Anderson, Nat. Mater. 2002, 1, 160 – 164.
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