Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

•Water-soluble hydrazone photoswitch templates the enzymatic synthesis of γ-cyclodextrin•The hydrazone template improves the selectivity and yield of γ-CD production•The Z hydrazone isomer binds strongly to γ-CD while the E isomer has low affinity•The photoremovable template is a proof-of-principle strategy for efficient γ-CD isolation Cyclodextrins (CDs) are extensively used as hosts for (bio)active molecules in the food, cosmetic, and pharmaceutical industries. Out of the three commonly used CDs, α-, β-, and γ-CD, the latter is the largest and most water soluble and thus can best encapsulate and solubilize bulky guests, such as vitamins and sensitive dyes. The encapsulation of drugs with γ-CD can provide stabilization against autoxidation during storage and allow for lower drug doses, thus mitigating irritation and toxicity. The high water solubility of γ-CD, however, complicates its isolation during industrial production, necessitating the use of an energy-consuming and cost-increasing distillation process. We show how a hydrazone photoswitch template can be used in the enzymatic production of CDs to specifically favor the formation of γ-CD. The template can then be removed from inside the γ-CD by photoirradiation, enabling product isolation processes that are more cost effective and environmentally friendly. The high water solubility of γ-cyclodextrin (γ-CD) lowers its synthetic yield and complicates its post-synthetic recovery, thus increasing production costs. Here, we report the use of a photoremovable hydrazone template as a proof-of-concept strategy for increasing the efficiency of the enzymatic synthesis of γ-CD and lowering the associated production costs. Our results show that while both the Z and E isomers of the hydrazone switch form low affinity (K = 250–725 M−1) complexes with β-CD (1:1), only the Z isomer of the switch can be included in γ-CD (2:1; K2 = 8,970 M−1). We used this property to preferably synthesize γ-CD and increase its yield by 6-fold and then took advantage of the photoremovable nature of the template to isolate the product. Considering the very limited number of photoswitches that can bind to γ-CDs, we anticipate that this newly discovered host-guest couple will open the way for designing γ-CD-based adaptive materials. The high water solubility of γ-cyclodextrin (γ-CD) lowers its synthetic yield and complicates its post-synthetic recovery, thus increasing production costs. Here, we report the use of a photoremovable hydrazone template as a proof-of-concept strategy for increasing the efficiency of the enzymatic synthesis of γ-CD and lowering the associated production costs. Our results show that while both the Z and E isomers of the hydrazone switch form low affinity (K = 250–725 M−1) complexes with β-CD (1:1), only the Z isomer of the switch can be included in γ-CD (2:1; K2 = 8,970 M−1). We used this property to preferably synthesize γ-CD and increase its yield by 6-fold and then took advantage of the photoremovable nature of the template to isolate the product. Considering the very limited number of photoswitches that can bind to γ-CDs, we anticipate that this newly discovered host-guest couple will open the way for designing γ-CD-based adaptive materials. IntroductionThe hydrophobic cavity and water solubility of cyclodextrins (CDs) have made them some of the most ubiquitous host molecules used in supramolecular chemistry.1Wenz G. Cyclodextrins as building blocks for supramolecular structures and functional units.Angew. Chem. Int. Ed. Engl. 1994; 33: 803-822https://doi.org/10.1002/anie.199408031Crossref Scopus (1358) Google Scholar, 2Szejtli J. 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Addressing the synthetic and isolation challenges facing the production of γ-CD requires a paradigm shift in approach, which will require the development of new photochromic guests.40Harris J.D. Moran M.J. Aprahamian I. New molecular switch architectures.Proc. Natl. Acad. Sci. USA. 2018; 115: 9414-9422https://doi.org/10.1073/pnas.1714499115Crossref PubMed Scopus (133) Google ScholarRecently,41Qian H. Pramanik S. Aprahamian I. Photochromic hydrazone switches with extremely long thermal half-lives.J. Am. Chem. Soc. 2017; 139: 9140-9143https://doi.org/10.1021/jacs.7b04993Crossref PubMed Scopus (85) Google Scholar, 42Shao B. Aprahamian I. Planarization-induced activation wavelength Red-Shift and thermal half-life acceleration in hydrazone photoswitches.ChemistryOpen. 2020; 9: 191-194https://doi.org/10.1002/open.201900340Crossref PubMed Scopus (7) Google Scholar, 43Shao B. Aprahamian I. 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Soc. 2019; 141: 8364-8371https://doi.org/10.1021/jacs.9b03932Crossref PubMed Scopus (59) Google Scholar that reversibly photoisomerize in organic solutions, the solid state, and aqueous media. Our interest in expanding the scope of photoswitches that can work in water, and act as guests to water-soluble macrocyclic hosts such as CDs, led us to the development of the first fully water-soluble hydrazone switch 1 (Figures 1 and S10). Here, we describe the photoswitching of 1 in pure water, and its complexation properties with β- and γ-CDs. While both isomers of 1 form inclusion complexes with β-CD (1:1), only 1-Z binds to γ-CD (2:1), allowing for optical control over the complexation/decomplexation process. We took advantage of this process and used it in the light-controlled templated enzymatic synthesis of γ-CD, which has never been demonstrated before. Moreover, we used the fact that γ-CD does not bind to the E isomer of the switch to photo-remove the template from the host, and thus isolate the synthesized γ-CD. This new photoswitchable host/guest couple not only presents a proof-of-concept strategy for improving the yields of γ-CD production, but it also opens the way for designing new γ-CD-based adaptive self-assembled materials.Results and discussionPhotoisomerization of the water-soluble hydrazoneThe target hydrazone (1; Scheme S1) was synthesized in a straightforward manner by reacting ethyl benzoylformate with 4-hydrazinobenzenesulfonic acid under reflux in water (78% yield). Both the E and Z isomers were separated using reversed-phase chromatography and characterized using NMR spectroscopy (Figures S1–S9) and high-resolution mass spectrometry.The switching of 1 (Figure 1) in water was first studied using UV-vis spectroscopy. The absorption spectrum of 1-Z (equilibrated in the dark) shows a band with a maximum (λmax) at 357 nm. Irradiating the solution with 410 nm light results in a shift of the λmax to 338 nm (Figure S10A). The photoisomerization efficiency of 1 was studied using 1H NMR spectroscopy (Figure S11). Upon 410 nm light irradiation, a sample of 1-Z yields a PSS410 consisting of 92% of the E isomer. The quantum yield (ΦZ→E) of the process was determined to be 2.6% ± 0.2% (Figure S12). Irradiation of the obtained sample with 340 nm light yields a PSS340 consisting of 65% of the Z isomer with ΦE→Z of 5.3% ± 0.3% (Figure S13). The switching process can be repeated ten times without any sign of degradation (Figure S10B). The study of the thermal half-life was not carried out because ester hydrolysis occurs at elevated temperatures in water. Nonetheless, the system is bistable as no signs of thermal isomerization of 1-E are detected by 1H NMR spectroscopy when stored in D2O, in the dark, and at ambient temperature for at least 4 months (Figure S14).Host-guest interaction between the hydrazone and cyclodextrinsNext, we studied the host-guest interaction of 1 with β-CD. NMR titrations were carried out by adding the CD into a D2O solution of 1-Z, resulting in slight upfield shifts for the CD proton signals, while mixed shifts were observed for the phenyl protons of 1-Z (Tables S1 and S2). The presence of only one set of signals in the 1H NMR spectra (Figure 2B) indicates that 1-Z and β-CD are in a rapid dynamic inclusion/exclusion exchange equilibrium on the NMR timescale. The result of a Job’s continuous variation analysis (Figure S18) indicated that the host and guest form a 1:1 complex, which was confirmed by ESI-MS measurements (Figure S19). Rotating frame nuclear Overhauser effect spectroscopy (ROESY) was used to elucidate the structure of the complex and rotating frame nuclear Overhauser effect (ROE) signals (Figure S35) were observed between the phenyl protons of 1-Z and protons H3′ and H5′ (Figure 2A) inside the β-CD cavity. These interactions suggest that 1-Z is encapsulated inside the β-CD through the stator phenyl group (Figure 1, shown in blue).Figure 2Host-guest interaction of 1 with β- and γ-CDsShow full caption(A) A Schematic representation of the cyclodextrins.(B and C) Inclusion and switching of 1 in (B) β-CD and (C) γ-CD as monitored by 1H NMR spectroscopy (D2O at 298 K). The spectra mainly focus on the CD signals as these show the clearest shifts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Photoisomerization with 410 nm light slightly shifts all the CD proton signals back to low field (Figure 2B), especially proton H5′. Similar chemical shifts were obtained when mixing pure 1-E with β-CD (Figure S17), indicating that the shifts observed upon photoisomerization do not originate from the small amount of leftover 1-Z at PSS410. These experiments indicate that both isomers of 1 bind to β-CD. To get a better appreciation of the binding affinities of the two isomers with the CD, isothermal titration calorimetry (ITC) was used (Table 1).47The binding of both isomers with α-CD was also studied using ITC. 1-Z has a very low affinity to α-CD (72 ± 1 M−1), while the binding affinity of 1-E was too low to be measured (Figures S32 and S33). Further characterization was not carried out. The binding constant between 1-Z and β-CD was calculated to be 725 ± 1 M−1 (Figure S21A) based on three consecutive measurements, while the binding constant between 1-E and β-CD was calculated to be 250 ± 25 M−1 under the same conditions (Figure S21B). Similar values were obtained from 1H NMR titrations,48Thordarson P. Determining association constants from titration experiments in supramolecular chemistry.Chem. Soc. Rev. 2011; 40: 1305-1323https://doi.org/10.1039/C0CS00062KCrossref PubMed Google Scholar, 49Hibbert D.B. Thordarson P. The death of the Job plot, transparency, open science and online tools, uncertainty estimation methods and other developments in supramolecular chemistry data analysis.Chem. Commun. 2016; 52: 12792-12805https://doi.org/10.1039/C6CC03888CCrossref PubMed Google Scholar, 50Thordarson P. (2015). http://supramolecular.org.Google Scholar 835 ± 15 and 205 ± 5 M−1 for 1-Z and 1-E, respectively (Figures S23 and S25).Table 1Summary of the inclusion behavior of 1 in CDs (25°C in water)Host/guestStoichiometryITC binding constants/M−1β-CD/1-Z1: 1725 ± 1β-CD/1-E1: 1250 ± 25γ-CD/1-Z1: 1320 ± 40 (K1)1: 28,970 ± 480 (K2)γ-CD/1-Ebinding affinity is too low to be measured Open table in a new tab We also studied the interaction of 1 with γ-CD. The proton signals of γ-CD shifted upfield and were broadened upon mixing 1-Z with γ-CD, indicating an interaction between the two (Figure 2C). The results of a Job’s plot analysis showed the formation of a 2:1 complex between 1-Z and γ-CD (Figure S26), and this stoichiometry was further confirmed by ESI-MS (Figure S27). ITC measurements (Table 1) yielded a binding constant of 320 ± 40 M−1 (K1) and 8,970 ± 480 M−1 (K2) between 1-Z and γ-CD using the sequential binding model (Figure S31A). Similar values were obtained from 1H NMR titrations (825 ± 285 and 8,205 ± 2,080 M−1; Figure S29), while ROESY measurements (Figure S40) indicated that a head-to-tail orientation of the hydrazone inside the cavity of the γ-CD could be the most likely structure of the complex.To obtain further information about the nature of the complex between 1-Z with γ-CD, structure refinement was carried out using molecular dynamics (MD) simulations. The initial structure was generated by a metric matrix distance geometry program,51Havel T.F. An evaluation of computational strategies for use in the determination of protein structure from distance constraints obtained by nuclear magnetic resonance.Prog. Biophys. Mol. Biol. 1991; 56: 43-78https://doi.org/10.1016/0079-6107(91)90007-fCrossref PubMed Google Scholar which utilizes the distances generated from the geometry of the molecules (holonomeric matrix) and the ROEs. Because of the high symmetry of the γ-CD, the ROE distances were treated as an ensemble (i.e., penalty was applied only if all possible combinations of the specific protons were in violation). The resulting structure was solvated with TIP4 water, energy minimized, first with the complex held rigidly, and then 100 ps of MD run at 300 K using NAMD (nanoscale molecular dynamics).52Phillips J.C. Braun R. Wang W. Gumbart J. Tajkhorshid E. Villa E. Chipot C. Skeel R.D. Kalé L. Schulten K. Scalable molecular dynamics with NAMD.J. Comput. Chem. 2005; 26: 1781-1802https://doi.org/10.1002/jcc.20289Crossref PubMed Scopus (12926) Google Scholar The structure obtained from the simulations agrees with the one predicted by the ROESY measurements (Figure S40), i.e., two hydrazones are encapsulated in a head-to-tail manner inside the γ-CD (Figure 3).Figure 3Proposed structure of (1-Z)2@γ-CD based on MD simulationsShow full captionThe side and top views of the simulated 2:1 complex between 1-Z and γ-CD are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Photoisomerization of 1-Z upon 410 nm light irradiation shifts the proton signals of γ-CD back to where they were originally without the addition of the hydrazone, indicating minimal interaction between γ-CD and the E isomer (Figure 2C). A similar result is obtained when pure 1-E is added to γ-CD (Figure S30). Moreover, no significant heat is released during ITC measurements when 1-E is added to γ-CD (Figure S31B). These results indicate that photoisomerization results in the exclusion of the hydrazone from the cavity. This process is reversible as irradiation with 340 nm light results in the formation of the Z isomer and upfield shift of the 1H NMR γ-CD signals.Hydrazone-templated enzymatic synthesis of cyclodextrinsConsidering the strong and selective molecular recognition of 1-Z by γ-CD, we sought to use 1 as a photoresponsive template for the cyclodextrin glucanotransferase (CGTase)-mediated enzymatic synthesis of γ-CD.53This cheaply available food-grade enzyme is from the same family of enzymes currently used in the manufacturing of CDs.Google Scholar CGTase can render the glycosidic linkages in α-1,4-glucans labile by catalyzing reversible transglycosylation and slow hydrolysis to generate dynamic mixtures of interconverting CDs and linear α-1,4-glucans. We have previously shown that