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Indirect Detection of Glycosidases using Amperometry Bharat Pralhadrao Gurale, Abasaheb Dhawane, Xikai Cui, Amrita Das, Xiaohu Zhang, and Suri S. Iyer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03943 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016

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Analytical Chemistry

Indirect Detection of Glycosidases using Amperometry Bharat P. Gurale†, Abasaheb N. Dhawane†, Xikai Cui, Amrita Das, Xiaohu Zhang, Suri S. Iyer * 788 Petit Science Center, Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA - 30302. Email: [email protected] Fax: 404-413-5505

†

These authors contributed equally to this work.

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Abstract: Glycosidases are essential enzymes that cleave glycoside bonds. The presence of glycosidases have been widely used to detect pathogens, label cells/tissues and report specific diseases. We have developed a rapid electrochemical assay to detect glycosidases. Exposure of electrochemically inactive substrates to glycosidases releases glucose, which can be measured easily using an electrochemical cell. Five different glycosidases were detected rapidly within 1 hour using disposable electrodes. This assay could readily be incorporated into repurposed glucose meters to rapidly detect glycosidases, which in turn could be useful to report the presence of a pathogen or illness.


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Introduction Glycosidases are important enzymes that degrade oligosaccharides and the glycan components of glycoproteins and glycolipids. These ubiquitous enzymes catalyze the cleavage of glycoside bonds and have been widely used in a number of industrial applications, such as food processing, wood processing for paper and pulp products.1-3 They have also been used as catalysts to form glycoside bonds via transglycosylation or by reverse hydrolysis.4 Numerous bacteria, fungi, parasites and viruses, express glycosidases to degrade glyco oligo/polymers. Glycosidases are also excreted in bodily fluids. Detection of glycosidases is often used to report presence/absence of a pathogen or indicate disease states. For example, detection of coliforms in drinking water, recreational pools and other water bodies is performed by testing for βgalactosidase and/or β-glucuronidase. 5,6 In a different study, α-mannosidase has been used as a biomarker to identify Chlamydia trachomatis infections.7 A comprehensive list of glycosidases used to detect specific pathogens is provided in a recent report. 8 Several colorimetric, fluorogenic and chemiluminescence substrates for glycosidases are commercially available for the rapid detection of pathogens and to report human disorders. Cleavage of the substrate releases the reporter molecule and the enzyme activity can be accurately measured using a fluorescence detector. For example, β-galactosidase cleaves 5bromo-4- chloro-3-indolyl-α-D-galactopyranoside9 to release galactose and 5-bromo-4-chloro3-hydroxyindole. The latter spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'dichloro-indigo, an intensely blue insoluble product.10 (Figure 1a) Since X-Gal itself is colorless, the presence and concentration of the blue-colored product can be used as a test for the presence of an active β-galactosidase. In a specific application, X-gal was used to differentiate endogenous β-galactosidase activity from lacZ gene expression.9 We were interested in developing an electrochemical assay that would use existing, ubiquitous glucose meters for the rapid detection of glycosidase activity. Unlike colorimetric methods that rely on a color change, which could be subject to misinterpretation especially when opaque and turbid samples are used, amperometric approaches are not subject to visual change in color. The use of glucose meters is very attractive for point of care diagnostics because glucose meters meets the ASSURED (Affordable, Sensitive, Selective, User-friendly, Rapid and robust, Equipment free and Deliverable to end-users) guidelines, established by the World Health Organization.11,12 Glucose meters are very affordable as the meters are a one time cost and the disposable strips are inexpensive. The system is highly selective as it uses glucose oxidase and measurements are performed using microliters of blood. Most personal glucose instruments are quite sensitive, with an operating detection range between 50-180 mg/dl and meet ISO accuracy standards, which requires accuracy to be within 15% of the actual number. The more recent meters and disposable strips, such as StatStrip glucose meters produced by Nova Biomedical are extremely accurate and have been approved by FDA for detecting glucose in neonatal and pediatric patients.13-15 In terms of ease of use, the test kits are very easy to use and are being used by millions worldwide. The tests are also very rapid with results obtained within seconds and the shelf life of most disposable strips is six months from the manufacturing date. Unlike PCRs, the device is equipment free, uses a battery as an electrical source and the results can be downloaded onto a personal computer or mobile device. Finally, the glucose 3 ACS Paragon Plus Environment


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meter is portable, lightweight and has a small footprint making it readily deliverable to endusers. Taken together, glucose meter represent a very important technological advance that has helped millions of patients to monitor their glucose and control complications related to diabetes mellitus. In this report, we have developed glycoside specific substrates that release glucose upon exposure to their respective glycosidases.

Experimental Section A. Electrochemical system: The three electrode electrochemical cell was developed as described previously.16,17 B. Detection of Glycosidases: All enzymes were incubated with their respective substrates (100 µL, 2 mM in DI water) at 37 oC for 1 h. 20 µL of the solution was used to detect the current using the printed electrode system. For α-mannosidase, a solution of the enzyme (100 µL, 10 U mL-1 in 0.05 M Citrate buffer, pH 4.5) was added to a solution of A. For β-galactosidase, a solution of the enzyme (100 µL, 100 U mL-1 in PBS buffer, pH 7.4) was added to a solution of C. For β-glucuronidase , a solution of the enzyme (100 µL, 15000 U mL-1 in phosphate buffer, pH 6.8) was added to a solution of D. For βN-acetylglucosaminidase, a solution of the enzyme (100 µL, 10 U mL-1 in 0.05 M citrate buffer, pH 5.0) was added to a solution of F. For α-L-Fucosidase, a solution of the enzyme (100 µL, 10 U mL-1 in 0.05 M citrate buffer, pH 5.5) was added to a solution of G or H. C. Limit of Detection studies: A solution of α-mannosidase (50 µL, 10-0.01 U mL-1 in 0.05 M Citrate buffer, pH 4.5) was added to a solution of A (50 µL, 4 mM in DI water). The mixture was incubated at 37 oC for 1 h. 20 µL mixture was used to detect the current using the printed electrode system. D. Time course studies: A solution of α-mannosidase (250 µL, 10 U mL-1, 0.5 U mL-1 or 0.1 U mL-1 in 0.05 M Citrate buffer, pH 4.5) was added to a solution of A (250 µL, 2 mM in DI water). The mixture was incubated at 37 oC. 20 µL of the solution was taken to detect the current using the printed electrode system at 15 min, 30 min, and 60 min respectively.

Results and Discussion Our approach to the detection of glycosidases was to design and develop compounds that could release glucose upon action of enzymes. The released glucose can be measured directly using glucose meters. (Figure 1b) The work flow of this assay is depicted in Figure 1c. There are two components of the assay. In the first part, the sample is incubated with the substrate and glucose is released if the enzyme is present. The second part involves detection of glucose. Briefly, a drop of the sample is tested with screen printed electrodes impregnated with glucose oxidase and a mediator. In this study, we purchased screen printed electrodes with a 3 electrode configuration (CH instruments, Austin, TX). We deposited glucose oxidase and the mediator (Prussian blue) as described previously. 16,17 Glucose oxidase and the cofactor, FAD, present on the strip oxidizes glucose to gluconolactone and the cofactor gets reduced to FADH2. O2 4 ACS Paragon Plus Environment

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oxidizes the FADH2 back to FAD and hydrogen peroxide (H2O2) is released. H2O2 oxidizes Prussian white to Prussian blue, which is converted back to Prussian white via electron gain at a low potential. The gain/loss of electrons is measured by a potentiostat or a glucose meter. If glucose is present in the sample, it will indicate presence of the enzyme, which can be directly correlated to a disease state or presence of the pathogen. This approach has been used to detect pathogens, nucleic acids and enzymes.18-25 Recently, we reported the detection of a library of unique influenza viruses using this approach.17 Here, we report the design, synthesis of a panel of glycosidase specific substrates and their amperometric detection using disposable test strips. We synthesized glucose bearing substrates for five different glycosidases, namely α-Dmannosidase, β-D-galactosidase, β-D-glucuronidase, β-D-N-acetylglucosaminidase and α-Lfucosidase. The structures of the compounds for their respective enzymes are depicted in Table 1. Since the enzymes are highly specific for their respective substrates, the negative control for a particular enzyme is a compound that is specific for a different enzyme. The syntheses of the compounds are depicted in Scheme 1. Briefly, a suitably protected donor bearing a good leaving group at the anomeric position was reacted with 4-hydroxyl benzaldehyde, followed by reduction of the aldehyde group to the corresponding alcohol, which was reacted with a suitably protected glucose derivative to yield the coupled product. We choose to introduce the benzyloxy spacer between the two monosaccharides as it provides a degree of flexibility for the enzyme to bind and cleave the glycoside bond. Removal of the protecting groups results in the desired product. As an example, the synthesis of the substrate for α-D-mannosidase, (α-Dmannopyranosyloxy)-1-benzyloxy]-1-α/β-D-glucopyranoside is described. The known 2,3,4,6 tetraacetate α-D mannose trichloroacetimidate donor 126 and p-hydroxy benzaldehyde as acceptor was dissolved in DCM and cooled to 0 °C. Addition of TMSOTf as the promoter resulted in exclusive α product 7 in high yield. The aldehyde was reduced to the alcohol using sodium borohydride. This alcohol was reacted with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside trichloroacetimidate 12 in the presence of TMSOTf as promoter to yield the predominantly β product (α/β in a 1:2 ratio) in high yields. by TLC). Zemplén deprotection of the acetate groups resulted in the desired products, which was purified using size exclusion chromatography. The syntheses of the other derivatives were performed in a similar manner. For α-L-fucosidase, we synthesized an additional derivative, where we attached the glucose directly to α-L-fucose (Scheme S1) because the enzyme is not very active when a benzyloxy spacer is introduced as a spacer. (Scheme S2) The panel of compounds was tested with their respective enzymes for glucose release. For these studies, the released glucose was measured amperometrically using a standard 3 electrode system as described previously. 17 When α-mannosidase was introduced to 1 mM of its substrate, (α-D-mannopyranosyloxy)-1-benzyloxy]-1-α/β-D-glucopyranoside, cleavage of the mannose occurred within minutes to yield the benzyloxy intermediate, which degraded rapidly to release glucose and the quinone derivative. (Figure 1b) The resulting current at 60 min was ~81 x 10-8 amperes, which suggested that 75% of the reaction was completed in one hour as complete cleavage should result in a value of 120 x 10-8 amperes. We note that one could measure released glucose continuously using a continuous glucose monitor, we choose to measure glucose after 1 hour for our initial studies. A continuous glucose monitor would provide more information, 5 ACS Paragon Plus Environment


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similar to a HPLC-PED (Pulsed Electrochemical Detection), however, the cost and equipment needs of a continuous meter and/or HPLC instrument would negate the "affordable" and "equipment-free" parts of the ASSURED guidelines.27 We also note that, despite two monosaccharides mannose and glucose being released upon the action of the enzyme, we choose to detect glucose since glucose meters are widely available, but detectors for mannose are unavailable. The negative control used for α-mannosidase was (α-D-glucuronic acid)-1benzyloxy]-1-α/β-D-glucopyranoside and the value for this molecule was only ~ 12 x 10-8 amperes. This value is not zero because the screen printed electrodes that we modified using glucose oxidase and the mediator produced a residual current when PBS buffer is used, which is ~ 9 x 10-8 amperes. Glucose is released when the enzymes for the respective substrates are used. Glucose is not released when a different substrate is used. (Table 1). The current observed in Table 1 is different for individual enzymes because the cleavage rates are slower for certain enzymes, with α-D-mannosidase exhibiting the highest rate and β-D-N-Acetyl glucosaminidase and α-L-fucosidase exhibiting lower rates. In the case of L-fucosidase, the substrate with the benzyloxy spacer resulted in inefficient cleavage as the active site of the enzyme has a tyrosine that stacks up against the phenyl ring, thereby inhibiting the rate of cleavage. However, when glucose is attached directly to L-fucose as shown in Entry 6, Table 1, the rate is faster. One concern related to the practicality of this assay when we transition to human clinical samples is the presence of interferences such as other saccharides such as galactose, lactose, etc. Additionally, glucose and/or enzymes that could degrade natural substrates to produce glucose may be present in the sample. To avoid false positives or negatives, we decided to measure the presence of glucose in the sample before and after addition of the substrate. The difference between the two readings is expected to indicate the presence of the associated enzyme. As a first step, we purchased artificial urine (Innovating Science™ Artificial Urine FSE was purchased from Fisher Scientific, Pittsburgh, PA) and spiked it with different carbohydrates such as D-galactose, D-mannose and lactose. As seen in Figure 2, entries I (D-galactose), II (Dmannose) and III (lactose), ∆I is negligible, indicating that these sugars do not react with glucose oxidase or the mediator in the coated screen printed electrodes. Next, we spiked one of the enzymes, β-glucuronidase without its substrate (entry IV). ∆I for this sample was also negligible, indicating that β-glucuronidase does not interact with anything in the sample to produce glucose. When we added the substrate to the sample containing β-glucuronidase, (entry V) we observed glucose released, indicating that the enzyme present in the sample reacts with the substrate to release glucose. We also added 1 mM glucose to the samples containing Dgalactose, D-mannose and lactose respectively (entries VI, VII and VIII), and we observed that glucose oxidase and mediator in the screen printed electrodes reacts only with glucose. The results indicate the following: (1) Glucose oxidase in the test strips reacts only with glucose and not with closely related sugars such as D-galactose, etc. (2) The glycosidase reacts specifically with its substrate. (3) The difference in current before and after addition of the substrate indicates the presence/absence of the glycosidase. Since our long term goal is to detect glycosidases in environmental or clinical samples, we tested the limit and time course of detection for one of the glycosidases. The limit of detection using a higher substrate concentration was determined to be 0.01 U/ml in 1 hour. (Figure 2) Next, we performed time course studies. We found that 0.1 units of mannosidase can be detected using this assay in 15 minutes. (Figure 3) This is a distinct advantage of this 6 ACS Paragon Plus Environment

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biochemical assay since most end-users suggest a test-to-result time of less than 20 minutes. For example, recent publications that focused on the needs of end users that use point of care diagnostics for sexually transmitted diseases clearly stated that they would like a test-to-result time of less than 20 minutes as there is a social stigma attached to these diseases.28,29 We anticipate better performance of this biochemical assay as improved substrates that result in faster rates are developed.

Conclusions In summary, we have developed a panel of glycoconjugates bearing a glucose derivative. Glucose is released when the respective enzymes are introduced, which is measured amperometrically using a standard 3 electrode electrochemical cell. This assay can be integrated in existing glucose meters as a method to detect these glycosidases. We note that a limitation of the present study is that artificial urine samples were used. Human urine samples from patients are expected to be more complex as they may contain interferences that may interact with either the substrate or the enzymes. Further optimization in substrate design and assay conditions that emphasize enzyme-substrate interactions over other interactions will be required when we transition this work from the laboratory to the clinic, however, as a first step, this assay shows promise in glycosidase detection using ubiquitous glucose meters. Since glycosidase release in body fluids can be indicative of the presence of a pathogen and or a disorder, this assay could potentially be used to monitor human diseases in a point of care setting. Furthermore, multiple glycosidases or other enzymes can be detected rapidly using multiple substrates; this information could assist physicians in identifying the causative agent(s) causing the illness.

Acknowledgements We are grateful to NIH-NIAID (R01-AI089450) for funding.

Supporting Information General procedures, abbreviations, synthetic details and NMR spectra for all new compounds are provided. This material is available free of charge via the internet at http://pubs.acs.org.



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References (1) Contesini, F. J.; de Alencar Figueira, J.; Kawaguti, H. Y.; de Barros Fernandes, P. C.; de Oliveira Carvalho, P.; da Graca Nascimento, M.; Sato, H. H. International journal of molecular sciences 2013, 14, 1335-1369. (2) Hilden, L.; Johansson, G. Biotechnol Lett 2004, 26, 1683-1693. (3) Sathya, T. A.; Khan, M. Journal of food science 2014, 79, R2149-2156. (4) Zeuner, B.; Jers, C.; Mikkelsen, J. D.; Meyer, A. S. J Agric Food Chem 2014, 62, 9615-9631. (5) Maheux, A. F.; Dion-Dupont, V.; Bouchard, S.; Bisson, M. A.; Bergeron, M. G.; Rodriguez, M. J. Journal of water and health 2015, 13, 340-352. (6) Sicard, C.; Shek, N.; White, D.; Bowers, R. J.; Brown, R. S.; Brennan, J. D. Anal Bioanal Chem 2014, 406, 5395-5403. (7) Wang, Z. Y.; Fu, G. Y.; Wang, S. M.; Qin, D. C.; Wang, Z. Q.; Cui, J. BMC Infect Dis 2013, 13, 36. (8) Orenga, S.; James, A. L.; Manafi, M.; Perry, J. D.; Pincus, D. H. J Microbiol Methods 2009, 79, 139-155. (9) Weiss, D. J.; Liggitt, D.; Clark, J. G. Hum Gene Ther 1997, 8, 1545-1554. (10) Coleman, D. J.; Kuntz, D. A.; Venkatesan, M.; Cook, G. M.; Williamson, S. P.; Rose, D. R.; Naleway, J. J. Anal Biochem 2010, 399, 7-12. (11) UNICEF/UNDP/World Bank/WHOSpecial Programme for Research and Training in Tropical Diseases. Accessible quality-assured diagnostics: annual report 2009; World Health Organization2010. (12) Peeling, R. W.; Holmes, K. K.; Mabey, D.; Ronald, A. Sexually transmitted infections 2006, 82 Suppl 5, v1-6. (13) Karon, B. S.; Blanshan, C. T.; Deobald, G. R.; Wockenfus, A. M. Diabetes technology & therapeutics 2014, 16, 828-832. (14) Lockyer, M. G.; Fu, K.; Edwards, R. M.; Collymore, L.; Thomas, J.; Hill, T.; Devaraj, S. Clin Biochem 2014, 47, 840-843. (15) Kitsommart, R.; Ngerncham, S.; Wongsiridej, P.; Kolatat, T.; Jirapaet, K. S.; Paes, B. Eur J Pediatr 2013, 172, 1181-1186. (16) Wu, S.; Liu, G.; Li, P.; Liu, H.; Xu, H. Biosens Bioelectron 2012, 38, 289-294. (17) Zhang, X.; Dhawane, A. N.; Sweeney, J.; He, Y.; Vasireddi, M.; Iyer, S. S. Angew Chem Int Ed Engl 2015, 54, 5929-5932. 8 ACS Paragon Plus Environment

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(18) Wang, Q.; Wang, H.; Yang, X.; Wang, K.; Liu, R.; Li, Q.; Ou, J. Analyst 2015, 140, 11611165. (19) Chavali, R.; Gunda, N. S. K.; Naicker, S.; Mitra, S. K. Anal Methods-Uk 2014, 6, 6223-6227. (20) Mohapatra, H.; Phillips, S. T. Chem Commun (Camb) 2013, 49, 6134-6136. (21) Xiang, Y.; Lu, Y. Nature chemistry 2011, 3, 697-703. (22) Xiang, Y.; Lu, Y. Anal Chem 2012, 84, 1975-1980. (23) Xu, X. T.; Liang, K. Y.; Zeng, J. Y. Biosens Bioelectron 2015, 64, 671-675. (24) Zhang, J.; Xiang, Y.; Novak, D. E.; Hoganson, G. E.; Zhu, J.; Lu, Y. Chem Asian J 2015, 10, 2221-2227. (25) Chen, S.; Gan, N.; Zhang, H.; Hu, F.; Li, T.; Cui, H.; Cao, Y.; Jiang, Q. Anal Bioanal Chem 2015, 407, 2499-2507. (26) Free, P.; Hurley, C. A.; Kageyama, T.; Chain, B. M.; Tabor, A. B. Organic & Biomolecular Chemistry 2006, 4, 1817-1830. (27) Fleming, S. C.; Kynaston, J. A.; Laker, M. F.; Pearson, A. D.; Kapembwa, M. S.; Griffin, G. E. J Chromatogr 1993, 640, 293-297. (28) Rompalo, A. M.; Hsieh, Y. H.; Hogan, T.; Barnes, M.; Jett-Goheen, M.; Huppert, J. S.; Gaydos, C. A. Sexual health 2013, 10, 541-545. (29) Hsieh, Y. H.; Hogan, M. T.; Barnes, M.; Jett-Goheen, M.; Huppert, J.; Rompalo, A. M.; Gaydos, C. A. PLoS One 2010, 5, e14144.



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Figure and Scheme Captions: Figure 1. A. Colorimetric and B. electrochemical detection of glycosidases. C. Workflow of the assay that depicts the two components, (i) production of glucose and (ii) detection of glucose. Figure 2. Different compounds were spiked in Innovating Science™ Artificial Urine FSE (Fischer Scientific, Pittsburgh, PA, USA) as indicated in the entries and glucose was measured after 1 hour. (I) 1mM D-Galactose (II) 1mM D-Mannose (III) 1mM Lactose (IV) 15000 UmL-1 β-Glucuronidase (V) 1mM compound D was incubated with β-Glucuronidase 15000 UmL-1 at 37oC for 1 h. (VI) 1mM D-Galactose plus 1 mM D-glucose (VII) D-Mannose plus 1 mM Dglucose (VIII) Lactose plus 1 mM D-glucose. The current was measured in amperes after 100 s using an amperometric i-t curve at a working potential of 0.00 V. T he y axis, ∆I, represents the difference in current before and after addition of the reagent. All experiments were performed in triplicate independently on different days. Figure 3. Limit of detection studies. Compound A was incubated with different concentrations of α-mannosidase at 37 oC for 1 h. Inset: Expanded region from 0.0 U/ml to 1.0 U/ml. The current was measured in amperes after 100 s using an amperometric i-t curve at a working potential of 0.00 V. The y axis, ∆I, represents the difference in current between the negative control (no enzyme) and the sample. The x-axis represents different concentrations. All experiments were performed in triplicate independently on different days. Figure 4. Time course studies. Compound A was incubated with α-Mannosidase at 37 oC. The current was measured at 15 min, 30 min, and 60 min respectively. The current was measured in amperes after 100 s using an amperometric i-t curve at a working potential of 0.00 V. The y axis, ∆I, represents the difference in current between the negative control (no enzyme) and the sample. All experiments were performed in triplicate independently on different days. Scheme 1. Reagents and conditions: a) 6, CH2Cl2, TMSOTf, 0 oC, 1 h, 65-75%; b) 6, CH3CN, Ag2O, 12 h, 70%; c) CH2Cl2: MeOH (1:1), NaBH4, 0 oC to rt, 1 h, 75-80%; d) 12, CH2Cl2, TMSOTf, -20 oC to 0 oC, 1 h, 60-70%; e) Zn dust, CuSO4, Ac2O, AcOH, rt, 3h, 65%; f) Ethylenediamine, MeOH, reflux, 4h, 70%; g) MeOH, NaOMe, rt, 1 h, 71-76%; for D: LiOH, MeOH:H2O: THF (3:1:1), rt, 1 h, 69%. Table 1. Electrochemical detection of glycosidases measured by electrochemical assay. 100 µL of glycosidase was mixed with 100 µL of buffer with respective compounds for 1 h at 37 0C. Glucose concentration was determined using 20 µL of this solution.


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Figure 1a

Figure 1b

75 mg/ml

Figure 1c



Figure 2

60

-8

∆ I/ 10 A

80

40 20 0

I

II III IV V VI VII VIII

Concentration/ UmL

-1

Figure 3

Figure 4

80 60

0.1 U/mL 0.5 U/mL 10 U/mL

-8

∆ I/ 10 A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40 20 0

5 10 15 -1 Concentration/ UmL 12 ACS Paragon Plus Environment

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Scheme 1. R4 R1 Y O R3 AcO R2 X

R4Y R1 O R3 AcO R2 O

a, c for 7, 8, 10, 11 b, c for 9

OH

X = OC(NH)CCl3 except for 3 X = Br HO

AcO AcO AcO

CHO

12

6 HO HO HO R4 Y

R3 HO

R1 O

O

NH

OAc O

AcO AcO AcO

O

g

HO O

d CCl3

R4 Y

for D, h

R2 O

R3 AcO

R1 O

O

AcO O

R2 O

e f



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Table 1. Entry

Enzyme

1

α-Mannosidase

2

β-Galactosidase

Current (x 10-8) A

Substrate

81.0+1.8

Negative Control

Current (x 10-8) A

12.3+1.2

10.2+0.6 50.9+1.7

3

β-Glucuronidase

35.8+1.1

10.4+1.3

4

β-N-Acetyl glucosaminidase

28.5+4.0

11.1+2.3

5

24.5+3.2 α-L-Fucosidase

9.7+3.5 44.5+2.9

6


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For TOC only

75 mg/ml


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