Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Photoswitchable Heparinase III for Enzymatic Preparation of Low Molecular Weight Heparin Yayun Gu,‡ Xuri Wu,‡ Huan Liu, Qi Pan, and Yijun Chen* State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, 24 Tongjia St., Nanjing, Jiangsu Province 210009, People’s Republic of China S Supporting Information *
ABSTRACT: A photocontrollable biocatalyst was rationally designed by site-specifically linking a photoswitchable azobenzene derivative to the only cysteine residue in heparinase III mutant (K130C). Upon photoswitch, the enzymatic degradation of heparin could be artificially controlled to produce low molecular weight heparin with more uniform molecular weight and an increase in anticoagulant activity.
U
enzymes requires more sophisticated design and careful choice of the site for the linkage because the activity of an enzyme by incorporating azobenzene moiety is extremely sensitive to the selection of pivotal residues.8 Usually, the coupling site far away from active site is unable to trigger the control of enzymatic activity. However, if the coupling site is too close to the active pocket, enzymatic activity may largely be influenced or even lost, resulting in only very few successful examples to date.8,9 Thus, the choice on the coupling site in an enzyme is particularly critical to the design and construction of a photoswitchable biocatalyst. According to the crystal structure of heparinase III from Pedobacter heparinus (PhHepIII),10 the site of Lys130 is adjacent to the active pocket and presented at the surface of the substrate entrance. Meanwhile, regardless of its structure of being a monomer or disulfide-linked homodimer, mutant K130C of PhHepIII was previously demonstrated to completely retain the activity on heparin degradation from the comparison of various mutants around the active site,11 which provides a unique opportunity for sitespecific coupling of the only cysteine residue with an azobenezene moiety for the regulation of its degradation activity to prepare LMWH. In the present study, a novel photoswitchable biocatalyst K130C−N,N-dimethylacrylamide-co-4-phenyl azophenyl acrylate copolymer (K130C−DMAA) was constructed by a covalent linkage between a polymeric azobenzene derivative and the only cysteine residue in mutant K130C of PhHepIII. With this designed photoswitchable HepIII, the allosteric azobenezene moiety was triggered by UV/vis switch to allow reversible
nfractionated heparin (UFH) is a mixture of linear, negatively charged and high molecular weight glycosaminoglycans to inhibit the activities of factor Xa and factor IIa in the coagulation cascade, and is widely used in clinical practice as an anticoagulant drug for nearly a century.1 Unfortunately, hemorrhage and heparin-induced thrombocytopenia are common adverse effects of UFH, which seriously increases the risks for its clinical use.1a Later, low molecular weight heparin (LWMH) with higher anticoagulant activity and lower incidences of side effects than UFH was prepared through various chemical and enzymatic depolymerization of heparin and gradually replaced UFH for clinical application.1b,2 Controlled enzymatic depolymerization of heparin is a feasible approach for the mass production of LMWH. Currently, enzymatic production of LMWH usually uses the combination of heparinase I/II/III from Pedobacter heparinus (also known as Flavobacterium heparinum) as biocatalysts. However, the process and outcomes are difficult to control.3a In the case of using single enzyme, heparinase III can selectively depolymerize heparin at undersulfated domains without destroying the antithrombin III binding site, and this enzyme is most likely to be a useful tool for the preparation of novel LMWH.3b Introduction of photoswitches for optically regulating the functions of various proteins and peptides, which can effectively probe the interactions and functions of biomacromolecules,4 has been proved highly valuable. Among different photosensitive molecules, azobenzene and its derivatives have served as excellent molecular photoswitches for the modulation of protein functions by cis/trans isomerization responding to different light irradiation or thermal relaxation.5−7 Different from other proteins, the coupling of photoswitches with © XXXX American Chemical Society
Received: October 26, 2017
A
DOI: 10.1021/acs.orglett.7b03340 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 1. Schematic illustration of photoswitchable K130C−DMAA conjugate for the enzymatic preparation of LMWH. K130C−DMAA represents the photoswitchable mutant K130C of PhHepIII. DMAA is the abbreviation of N,N-dimethylacrylamide-co-4-phenyl azophenyl acrylate copolymer.
control of the enzyme activity on heparin degradation. This controllable process showed simple, efficient, and precise control on the preparation of novel LMWH with more uniform molecular weight and easier product recovery (Figure 1). First, given that azobenzene photoswitches can be conveniently linked to proteins by selective and specific conjugation to the thiol group of cysteine residues via Michael addition,12 we synthesized photoresponsive DMAA and its vinyl sulfone (VS) derivative DMAA−VS according to the literature (Scheme S1).9 The azobenzene−polymer DMAA with molecular weight of 3702 Da and polydisperdity of 1.21 was prepared based on reversible addition fragment chain transfer polymerization under optimized conditions (Figure S1). To obtain photosensitive polymer with a terminal vinyl sulfone group for Michael addition, we then synthesized the derivative DMAA−VS for covalently linking thiol groups, and the structure of DMAA−VS was characterized by 1H NMR (Figure S2). Subsequently, the photoswitchable features of DMAA−VS were confirmed according to the literature (Figure S3).9,12 Next, covalent and specific coupling of the photopolymer DMAA−VS with mutant K130C was achieved by treating with a reducing agent Tris(2-carboxyethyl) phosphine hydrochloride. Based on the thermal-sensitive characteristics of DMAA−VS,9,12 90% of K130C−DMAA conjugate was successfully obtained from thermal-induced precipitation judged by 8% SDS-PAGE (Figure S4). To examine the optically allosteric control of K130C−DMAA conjugate, the photoswitchable behaviors of the conjugate solution were examined from UV exposure followed by blue light irradiation. As expected, wavelength-dependent switch of the conjugate was verified by UV/vis spectroscopy, showing increased absorptions at 325 nm (trans, blue) and 440 nm (cis, red) (Figure 2A). In addition, CD spectra of the conjugate indicated that the secondary structure of mutant K130C was not affected by the conjugation with the photosensitive polymer (Figure S5).
Figure 2. Characterization of photoswitchable K130C−DMAA conjugate. (A) UV−vis spectra of K130C−DMAA conjugate under different irradiations. (B) Illustration of the conformational change of K130C−DMAA conjugate. The cartoon structure of mutant K130C originated from the crystal structure of PhHepIII (PDB: 4MMH).
Together, the photosensitivity and structural features of K130C−DMAA conjugate provide a basis for the allosteric photocontrol of heparin degradation without affecting its structural architecture and catalytic activity. To investigate the precise control of enzymatic activity from the irradiation by a blue light and UV-LED, the cleaved fragments from heparin by photosensitive K130C−DMAA conjugate were analyzed and compared. As shown in Figure 2B, in accordance with the cis conformation of DMAA, oligosaccharide products from heparin by UV-irradiation (365 nm, 5 mW/cm2, 5 cm) of K130C−DMAA conjugate for 60 min were detected by DNS chromogenic method,11 which is comparable to the products produced by wild-type PhHepIII. The activity of K130C−DMAA conjugate in the active cis form for the enzymatic deploymerization of heparin (0.75 μg/min/ mg) was similar to that of wild-type HepIII (0.8 μg/min/mg) and mutant K130C (0.78 μg/min/mg). As expected, no detectable products were found from the inactive trans form of B
DOI: 10.1021/acs.orglett.7b03340 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. Molecular Weight and Anticoagulation Activity of Different LMWH Products parameters molecular weight
anticoagulation activity
Mwb (kDa) Mnc (kDa) Mw/Mn (PDI)d Mw < 8000 (%) anti-Xa (IU/mg) anti-IIa (IU/mg) anti-Xa/anti-IIa
heparin (substrate)
LMWH from photoswitched process
LMWH from PhHepIII
LMWH from mutant K130C
enoxaparina
34.40 23.80 1.45 0.25% 188.10 ± 1.13 187.20 ± 1.41 1.00
4.60 3.46 1.33 90.95% 165.80 ± 2.41 80.20 ± 0.59 2.07
8.43 4.76 1.77 58.71% 178.79 ± 2.19 140.42 ± 3.17 1.27
7.92 1.43 5.50 59.10% 173.63 ± 1.98 128.50 ± 1.43 1.35
5.04 4.03 1.25 69.23% 118.75 ± 1.42 30.84 ± 5.68 3.85
a Enoxaparin is a commercially available LMWH prepared by chemical depolymerization. bMw, weight-average molecular weight of low molecular weight heparin determined by gel permeation chromatography. cMn, number-average molecular weight of low molecular weight heparin determined by gel permeation chromatography. dPolydispersity index (PDI) represents molecular weight distribution. Data are average values of three independent experiments with standard deviations.
three cycles of photoswitchable degradation, approximately 80% of enzyme activity was retained (Figure S7D), which possesses certain advantages over conventional enzyme immobilization.5 To explore the potential utility of this enzymatic process, the anticoagulant activity of the LMWH products from this photoswitchable biocatalytic system was examined. The LWMH products degraded by wild-type PhHepIII maintained high anti-IIa activity (140.42 ± 3.17 IU/mg), consistent with reported values in the literature.3c On the contrary, the LMWH products by photoswitchable K130C−DMAA conjugate under UV-irradiation showed lower anti-IIa activity (80.20 ± 0.59 IU/ mg) with a significant increase of the ratio of anti-Xa/anti-IIa (Table 1). Although the anti-IIa activity of LMWH prepared from K130C−DMAA was still higher than that of FDAapproved enoxaparin (Table 1), further mechanistic study would offer a new opportunity for the improvement. In conclusion, the coupling of mutant K130C of PhHepIII with polymeric azobenzene resulted in a photoswitchable enzyme for heparin depolymerization. This recyclable enzyme was able to degrade heparin under UV-irradiation to precisely control the process of heparin degradation, which offers a novel approach for the preparation of LMWH with better quality. Meanwhile, the present study represents a new example on enzyme redesign and biocatalytic process development, which can facilitate the development and application of novel biocatalytic systems in the preparation of valuable pharmaceuticals and chemicals.
K130C−DMAA conjugate with blue light irradiation (470 nm), indicating that the activity of Hep III was precisely controlled by the cis/trans isomerization of the polymeric azobenzene to exhibit an on/off switch (Figure S6). To optimize the reaction conditions for K130C−DMAA conjugate in heparin depolymerization, UV 365 nm, 5 mW/cm2 of light intensity, 5 cm of irradiation distance, and 60 min of total UV illumination divided by 15 min continuous UV irradiation (“switch on”) and 15 min visible light irradiation (“switch off”) were found to be the best (Tables S1−S3). Then, under the optimal conditions, heparin was photoswitchably depolymerized by K130C−DMAA conjugate to generate LMWH fragments at a 10 mL scale. The resulting oligosaccharide products were obtained by thermal-induced protein removal, desalting, and lyophilization. After the exclusion of residual proteins in the lyophilized samples, molecular weight and homogeneity of LMWH products from photoswitchable K130C−DMAA conjugate, mutant K130C, and wild-type PhHepIII were analyzed and compared by gel permeation chromatography. As shown in Table 1, commercial enoxaparin prepared by chemical degradation of heparin displayed the best polydispersity index, but the switchable process using K130C−DMAA conjugate could produce LMWH with lower and more uniform molecular weight (Mw: 4600 Da; PDI: 1.33), compared to wild-type PhHepIII (Mw: 8430 Da; PDI: 1.77) and mutant K130C (Mw: 7920 Da; PDI: 5.50). Thus, the photocontrolled enzymatic process resulted in the decrease of molecular weight and the enrichment of sulfated active fragments for LMWH products according to the literature.13 Although the cleavage sites and mechanism are unknown, it could be either a synergetic effect from enzymatic degradation and photoactivation or the increase of accessibility of the substrate to the active site, which remains for further investigation. The stability and reusability of the biocatalyst are important measures for the utility of a biocatalytic process. After storing at 4 and 25 °C, the half-lives of the photoswitchable biocatalyst were 8.9 and 5.1 days (Figure S7A). The thermal-induced precipitation of K130C−DMAA only occurred at temperatures above 40 °C. Meanwhile, greater than 75% of total activity for the pellets of K130C−DMAA conjugate was observed after incubating at 40 °C for 20 min (Figure S7B,C), suggesting that 40 °C could be used as a temperature for the precipitation of the biocatalyst. With regard to the reusability, the photoswitchable biocatalyst was efficiently recovered after thermalinduced precipitation, which easily separates the biocatalyst from LMWH products in the aqueous reaction media. After
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03340. Experimental details and characterization data for K130C−DMAA conjugate and LMWH (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] ORCID
Yijun Chen: 0000-0002-4920-152X Author Contributions ‡
Y.G. and X.W. contributed equally to this work.
C
DOI: 10.1021/acs.orglett.7b03340 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China (No. 111-2-07), the National Natural Science Foundation of China (No. 21778076), and the PAPD of Jiangsu Province.
■
REFERENCES
(1) (a) Onishi, A.; St Ange, K.; Dordick, J. S.; Linhardt, R. J. Front. Biosci., Landmark Ed. 2016, 21, 1372−1392. (b) Oduah, E. I.; Linhardt, R. J.; Sharfstein, S. T. Pharmaceuticals 2016, 9, 38−50. (2) Harenberg, J. Int. J. Cardiol. 2016, 212, S10−S13. (3) (a) Wu, J.; Zhang, C.; Mei, X.; Li, Y.; Xing, X. H. Carbohydr. Polym. 2014, 101, 484−492. (b) Xiao, Z. P.; Tappen, B. R.; Ly, M.; Zhao, W. J.; Canova, L. P.; Guan, H.; Linhardt, R. J. J. Med. Chem. 2011, 54, 603−610. (4) Broichhagen, J.; Frank, J. A.; Trauner, D. Acc. Chem. Res. 2015, 48, 1947−1960. (5) Roy, I.; Sharma, S.; Gupta, M. N. Adv. Biochem. Eng./Biotechnol. 2004, 86, 159−189. (6) (a) Mart, R. J.; Allemann, R. K. Chem. Commun. 2016, 52, 12262−12277. (b) Szymański, W.; Beierle, J. M.; Kistemaker, H. A.; Velema, W. A.; Feringa, B. L. Chem. Rev. 2013, 113, 6114−6178. (c) Beharry, A. A.; Woolley, G. A. Chem. Soc. Rev. 2011, 40, 4422− 4437. (7) Roy, I.; Gupta, M. N. Chem. Biol. 2003, 10, 1161−1171. (8) (a) Yang, W. K.; Zhu, L. J.; Cui, Y. C.; Wang, H. W.; Wang, Y. W.; Yuan, L.; Chen, H. ACS Appl. Mater. Interfaces 2016, 8, 15967− 15974. (b) Korbus, M.; Backé, S.; Meyer-Almes, F. J. J. Mol. Recognit. 2015, 28, 201−209. (c) Horstmann, B.; Korbus, M.; Friedmann, T.; Wolff, C.; Thiele, C. M.; Meyer-Almes, F. J. Bioorg. Chem. 2014, 57, 155−161. (9) Shimoboji, T.; Larenas, E.; Fowler, T.; Kulkarni, S.; Hoffman, A. S.; Stayton, P. S. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 16592−16596. (10) Hashimoto, W.; Maruyama, Y.; Nakamichi, Y.; Mikami, B.; Murata, K. Biochemistry 2014, 53, 777−786. (11) Gu, Y. Y.; Lu, M. L.; Wang, Z. Q.; Wu, X. R.; Chen, Y. J. Chem. Eur. J. 2017, 23, 2548−2551. (12) Thordarson, P.; Le Droumaguet, B.; Velonia, K. Appl. Microbiol. Biotechnol. 2006, 73, 243−254. (13) (a) Volpi, N.; Linhardt, R. J. Nat. Protoc. 2010, 5, 993−1004. (b) Hyun, Y. J.; Jung, I. H.; Kim, D. H. Carbohydr. Res. 2012, 359, 37− 43.
D
DOI: 10.1021/acs.orglett.7b03340 Org. Lett. XXXX, XXX, XXX−XXX
