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[email protected] Current Medicinal Chemistry, 2013, 20, 3797-3801
3797
Carbasugar Probes to Explore the Enzyme Binding Pocket at the Anomeric Position: Application to the Design of Golgi Mannosidase II Inhibitors M.V. Vinader and K. Afarinkia* Institute of Cancer Therapeutics, University of Bradford, Bradford, BD7 1DP, United Kingdom Abstract: A methodology is described for the highly efficient and divergent synthesis of pseudosugars which allows the stereoselective introduction of polar groups at either the or the pseudoanomeric positions. Using this method, a series of 3-deoxycarbasugar analogues of mannose bearing a pyridyl group are rationally designed, prepared and tested for inhibition of Golgi -mannosidase II.
Keywords: Carbasugar, Golgi mannosidase II, molecular modelling, synthesis. INTRODUCTION
a
Glycosyl hydrolases are an important class of enzymes that control many significant biological transformations, and are implicated in numerous pathophysiological events [1]. Therefore, chemical agents that can modulate the activity of these enzymes are of great interest, both as biological tools for understanding disease mechanisms, as well as potential therapeutic agents [2]. One of the most important classes of small molecule glycosyl hydrolase inhibitors are carbasugars, structural analogues of “true” sugars in which the ring (endocyclic) oxygen atom is replaced by a carbon atom [3]. Carbasugars provide a particularly interesting and straightforward approach to glycosyl hydrolase inhibition because they enable the rational design of specific inhibitors for a given enzyme, through mimicry of their substrate and an understanding of the mechanism of the hydrolysis (Fig. 1) [4]. It is postulated that because of their structural similarity to “true” sugars carbasugars bind within the active site of the enzymes, in competition with the enzyme’s natural substrate, resulting in the inhibition of the enzyme’s function. More recently, rational design of carbasugars has included the incorporation of an alkene function, to better mimic the structural distortions the natural substrate undergoes during the hydrolysis, as well as the introduction of polar groups in the pseudo-anomeric position. The rational for the latter is that cleavage of the anomeric link, in both inverting and retaining mechanisms, involve protonation of the anomeric oxygen and/or nucleophilic attack at the anomeric position by a carboxylic acid residue at the active site (Fig. 1). Therefore, polar functional groups that interact strongly with these polar residues at the active site can bind more tightly, and more effectively inhibit the enzyme. *Address correspondence to this author at the Institute of Cancer Therapeutics, University of Bradford, Bradford, BD7 1DP, United Kingdom; Tel: (+44) 1274235831; Fax: (+44) 1274233234; E-mail:
[email protected]. 1875-533X/13 $58.00+.00
b O
O O
O
NH2 H
H
N O
R
NH2
OH B
N
H B = Polar group
Fig. (1). (a) Inverting mechanism of glycoside hydrolysis; (b) Role of a pseudoanomeric polar group in the binding site of glycosidase enzymes.
In this context, concise and straight forward synthesis of carbasugars to rationally investigate the role of pseudoanomeric configuration is highly useful in providing information about the type of residues and environment around the catalytic site. In continuation of our earlier work for the development of diversity-oriented synthesis of pseudosugars [5, 6], we now wish to highlight a short and divergent method for the stereocontrolled synthesis of carbamannose analogues with polar groups at either the or anomeric position, as well as analogues in which the pseudoanomeric position is flat, compounds 1, 2 and 3 (Fig. 2). The methodology is general, and adaptable for stereoselective introduction of various groups at BOTH anomeric configurations. Furthermore, we demonstrate one application of such molecules and report the unexpected in vitro activity of one these analogues against an -mannosidase, and provide computational rationalisation to support a probable role for the pyridyl pseudoanomeric group in those targets. The methodology uses the highly versatile Diels-Alder cycloaddition of 2(H)pyran-2-ones on which we [5-7] and a number of other groups [8] have extensively published and used as an efficient means of carbasugar synthesis. [5, 6] Thus, 5-(2-pyridyl)-2(H)pyran-2-one 4 was prepared from 5bromo-2(H)-pyran-2-one [9] using Stille or Suzuki cou© 2013 Bentham Science Publishers
3798 Current Medicinal Chemistry, 2013, Vol. 20, No. 30
Vinader and Afarinkia
pling,[10] and underwent Diels-Alder cycloaddition with benzyl vinyl ether [11] or dimethylphenylsilyl vinyl ether to afford a mixture of the endo and exo cycloadducts 5 and 6 from which the former were separated by silica gel chromatography (Scheme 1). The configurations of the cycloadducts were confirmed by the well-established protocols, based on the observation of both the chemical shifts and coupling constants of protons in NMR, laid down by Tomisawa and Hongo [12], and Afarinkia and Posner [7]. HO HO
OH 1
OH
HO HO
Py
OH
HO HO
2
Py
3
Py
Py = 2-pyridyl
Fig. (2). Proposed targets.
Hydrogenation of the endo cycloadduct 5a at room temperature and atmospheric pressure over palladium on charcoal afforded a single diastereomeric product. Based on precedence [5, 13], the product was assigned the structure 7. Further evidence in support of the proposed structures was obtained from the observation of a W-coupling between H-7 and H-6exo (4J= 2.3 Hz) which necessitates the two hydrogens to be in the same face of the molecule. In these bicyclic lactones H-6exo is identified through the large size (~ 10 Hz) of its syn coupling to H-5exo according to the rules set by Afarinkia and Posner [7]. The hydride reduction of bicyclic lactone 7 to afford carbasugar analogue 8 followed by removal of the benzyl group using PdCl2 as hydrogenation catalyst yielded carbasugar 1 (Scheme 2). The ring opening of the bicyclic lactone 5a under basic conditions gave compound 9, without the migration of the double bond. We attribute the lack of migration to the fact that under reaction conditions, deprotonation adjacent to the carboxylate function, which is a prerequisite for the migration of double bond, cannot occur. Hydrogenation of 9 afforded carboxy carbasugar 10 with >20:1 selectivity (Scheme 2). Reduction of carboxyl function, to afford 11, followed by hydrogenation catalysed by PdCl2 afforded desired compound 2. The difference in the selectivity of the hydrogenation of bicyclic cycloadduct and its ring opened derivative is explained in terms of steric encumbrance in the approach of the alkene moiety to the surface of hydrogenation catalyst. In the
cycloadduct 5a, the approach is from the same face as the bridging lactone due to the steric bulk of OBn substituent (Fig. 3) [5, 13]. However, the ring opened cycloadduct 9 adopts a pseudo boat conformation and the approach is from the face containing tertiary hydrogens, which even though are in axial orientation, are still less sterically demanding than the CH2CH(OR) bridge (Fig. 3). This rationale has been previously shown to be also applicable in the synthesis of phenantridone natural products [13]. We expected that hydride reduction of cycloadduct 5b would afford compound 12. Surprisingly however, the reaction affords the expected compound only as a minor product, with the majority of the product being dihydro compound 13 (Scheme 3). This is in contrast to the observation during hydride reduction of the hydrogenated cycloadduct 7 (Scheme 2). We attributed this difference to the chelation of the pyridyl ring to aluminum and direct delivery of the hydride reducing agent (Scheme 4). Nevertheless, removal of the silyl group from 12 afforded the desired compound 3 (Scheme 3). A similar sequence of reactions enabled the transformation of cycloadduct 14 to carbamannose analogues 15, 16 and 17, in which the anomeric substituents are phenyl rings rather than the pyridyl ring (Scheme 4). Unlike the hydride reduction of 5a, hydride reduction of 14 did not afford any dehydro analogues confirming the role of pyridyl nitrogen in directing the delivery of the hydride. We decided to test our molecules against Golgi mannosidase II (GMII). GMII is a key enzyme in the remodeling of a cell’s glycoprotein decoration [14] and has therefore been a target for therapeutic intervention in diseases such as cancer. Whilst a number of pseudosugars are know to inhibit the enzyme [15], no pyranose carbasugar analogue has been tested against it previously. We reasoned that carbamannose analogue 1, with a pyridyl group at the pseudoanomeric position, should be a good inhibitor of the mannosidase enzyme. Compounds 1-3 and 15-17 were tested against Jack bean (Canavalia ensiformis) GMII using an established procedure (Table 1) [16]. The alkaloid natural product Swainsonine 18 (Fig. 4), a known, clinically significant, highly potent GMII inhibitor [17], was also tested as control. No significant inhibition was detected for analogues 15, 16 and 17 up to 1 mM. However, as predicted by our hypothesis, compounds 1, was a moderately good inhibitor of the enzyme. The lack of activity for compound 15 confirms that the pyridyl group plays a critical role in the inhibition. O
O O
O
Br
O
i Py
O 4, 96%
O
O
OR
ii Py
Py = 2-pyridyl
OR 5a-c
Py
6a-c
R = OBn; 5a 54%; 6a 16% R = OSiMe2Ph; 5b 70%; 6b 22%
Reagents and conditions: i) Pd(PPh3)4, toluene, reflux, 20 h; ii) ROCH=CH2, sealed tube, 4.5 days
(Scheme 1). Preparation and cycloadditions of 5-pyridyl-2(H)-pyran-2-one.
Carbasugar Probes to Explore the Enzyme Binding Pocket at the Anomeric Position BnO
BnO
O
i
O N
100%
O
O H7exo
5a
N
H5exo H6exo
7
ii, 45%
iv, 74%
CH2OH
CO2H
OR1
OBn 1 N
OH
N 9
5 OH
8, R1 = Bn 1, R1 = H, 88%
iii i, 100%
CH2OH
CO2H
OR1
OBn ii 86%
OH
N 10
N
OH
11, R1 = Bn 2, R1 = H, 78%
iii
Reagents and conditions: i) H2, EtOAc, 10% Pd/C, 15 h, rt; ii) LiAlH4 (1.0 eq), THF, 20 h, rt; iii) H2, EtOH, PdCl2, 24 h, rt; iv) LiOH (1.2 eq), THF/H2O (5:1), 1 h, rt.
(Scheme 2). Preparation of compound 1, and 2. H CO2H RO
Sterically less hindered
Sterically less hindered
N
H
RO OH 9
O
N
O
5a
Fig. (3). Rationale for the selectivity in the hydrogenation step.
Interestingly, compound 3 was shown to be slightly less potent inhibitor than compound 1. This could be rationalized in terms of the similarity in the two compounds for the position of the pyridyl ring within the enzyme active site. However, contrary to our expectations, the analogue of the beta anomer, compound 2, was also active, albeit less than 1 and 3. The lack of potency in compound 17, confirmed that the pyridyl group plays a role in accounting for the activity of compound 3. In view of the numerous crystal structures of Golgi mannosidase II (EC 3.2.1.114) which are available, and the adaptability of the systems to molecular modelling [18], we
Current Medicinal Chemistry, 2013, Vol. 20, No. 30 3799
felt that in silico studies may be able to provide an insight into these observations. Using crystal structures of GMII from Drosophila melanogaster (dGMII, pdb code 1R33, 1PS3 and 1HXK) [19-21], we carried out a number of in silico studies for the binding of molecules 1-3 [22]. In order to ensure that the lack of a hydroxyl function does not significantly affect the binding mode of the molecule, we also computationally investigated the binding of analogues 15-17 and 19-21 (Fig. 4). We found that the calculated binding energy mirrored the observed potency. Thus the calculated relative binding energy for the anomer, 1, was higher than that for the “flat” analogue, 3, by 0.37 Kcal/mol and by 0.47 Kcal/mol for the anomer, 2. As confirmed experimentally, the calculated binding energies for 15, 16 and 17 were all lower than the corresponding pyridyl analogues by at least 1.5 Kcal/mol. Closer examination of the binding interactions showed significant differences in the binding modes of the molecules that could explain these differences (Fig. 5). In all models, the 4-hydroxy moiety was involved in two hydrogen bonding interactions with Y727 as a donor and D472 as an acceptor. The catalytic mechanism involves the R228 and D204, residues where the former abstract a hydrogen from the latter which is the nucleophilic residue [23]. In the models for both compounds 1 (the analogue of the alpha anomer) and compound 3, we observed an interaction between the pyridyl group and R228. This confirms the hypothesis that a polar group at the anomeric position of the carbasugar can indeed interact with the catalytic residues involved in the mechanism of the hydrolysis. As we had expected, we observed no interaction between the pyridyl group and R228 residues in compound 2 (the analogue of the beta anomer). Instead, we saw a strong chelation from the nitrogen of the pyridine ring to the zinc atom at the binding site (Figs. 5, 6). The models also suggest changes in the conformation of the molecule 2 to a twisted boat, that observation is entirely consistent with earlier evidence of significant flexibility for ligands within the binding pocket of dGMII [24]. Modeling studies therefore confirm that the original hypothesis was correct and a pyridyl group placed at the alpha anomeric position, as in compound 1, does in deed enable the molecules to bind more tightly at the active site and thus inhibit the enzyme. However, the experimental and computational studies also confirm that a polar group at the beta anomeric position can also provide binding interaction to the zinc atom at the active site. This weaker interaction can explain the unexpected potency of the beta anomer, 2. In continuation of this work, we are currently extending the scope of the methodology to prepare other probe molecules bearing different polar substituents in both the or anomeric positions. For example, we have prepared a range of 5-aryl-2(H)pyran-2-ones as a means of preparing a library of carbasugarrs to probe the active site of various glycosidase enzymes and will report these investigations in due course. In summary, we have demonstrated a concise and general method for the stereocontrolled preparation of analogues of mannose with a basic group at either the or anomeric
3800 Current Medicinal Chemistry, 2013, Vol. 20, No. 30
Vinader and Afarinkia
PhMe2SiO
CH2OH
CH2OH
OSiMe2Ph O
48%
O N
H
N
Al H
H
OH ii
i
5b
N
OH
OH 3
R = OH; 12, 16% R = H; 13, 69%
Reagents and conditions: i) LiAlH4 (1.0 eq), THF, 20 h, rt; ii) TBAF, THF, 5 minutes, rt.
(Scheme 3). Proposed mechanism for the hydride reduction of cycloadduct 5b and preparation of compound 3.
position, as well as molecules in which the pseudoanomeric position is flat. The methodology is general and can be applied to the introduction of a range of polar groups at the pseudo anomeric position of carbasugars. In addition, we have shown that the analogues can be used to probe the active site of a glycosyl hydrolase for binding interactions. CH2OH OH
i, ii O Ph OH
O
CH2OH
OAc
Ph 14
15
OH
ii Ph
CH2OH OH
i Ph
OH
16
OH
17
Fig. (5). Binding interactions at the active site of dGMII for (a) compound 1 and (b) compound 3.
Reagents and conditions: i) H2, EtOAc, 10% Pd/C, 15 h, rt; ii) LiAlH4 (1.0 eq), THF, 20 h, rt.
(Scheme 4). Preparation of compounds 15, 16 and 17. Table 1.
The IC50 Values of Swainsonine (As Reference) and Compounds 1, 2, 3 Against GMII from Jack Bean Compound
IC50 (n=3)
Swainsonine, 18
0.2±0.02 μM
1
40±10 μM
ACKNOWLEDGEMENTS
2
250±30 μM
3
50±5 μM
We thank Yorkshire Cancer Research and University of Bradford for financial support.
OH HO HO
N 18
HO HO HO
OH
HO HO HO
19 Py
OH
20
HO HO Py HO
Fig. (6). Binding mode of (a) compound 1, (b) compound 2, and (c) compound 3 at the active site of dGMII.
SUPPLEMENTARY MATERIALS
OH
21
Py
Fig. (4). Structure of compounds 18-21.
CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.
Supplementary material is available on the publisher’s web site along with the published article. Preparation and spectroscopic characterization of compounds and further details of the computational studies. REFERENCES [1]
(a) Ohtsubo, K.; Marth, J. D. Glycosylation in cellular mechanisms of health and disease. Cell, 2006, 126, 855-867. (b) Packer, N. H. ; Karlsson, N. G. Inhibition of glycosyltransferase activities as the basis for drug development. Methods Mol. Biol., 2009, 534, 359-
Carbasugar Probes to Explore the Enzyme Binding Pocket at the Anomeric Position
[2]
[3]
[4]
[5] [6]
[7]
[8]
[9] [10]
373. (c) Kajimoto, T.; Node, M. Inhibitors against glycosidases as medicines. Curr. Top. Med. Chem., 2009, 9, 13-33. Gloster, T. M. Development of inhibitors as research tools for carbohydrate-processing enzymes. Biochem. Soc. Trans., 2012, 40, 913-928. (b) Rempel, B. P.; Withers, S. G. Covalent inhibitors of glycosidases and their applications in biochemistry and biology. Glycobiology, 2008, 18, 570-586. (a) Cipolla, L.; Peri, F. Carbohydrate-based bioactive compounds for medicinal chemistry applications. Mini-Rev. Med. Chem., 2011, 11, 39-54. (b) Diaz, L.; Delgado, A. Medicinal chemistry of aminocyclitols. Curr. Med. Chem., 2010, 17, 2393-2418. (c) Cipolla, L.; La Ferla, B.; Airoldi, C.; Zona, C.; Orsato, A.; Shaikh, N.; Russo, L.; Nicotra, F. Carbohydrate mimetics and scaffolds: sweet spots in medicinal chemistry. Future Med. Chem., 2010, 2, 587599. (a) Vasella, A.; Davies, G. J. Böhm, M. Glycosidase mechanisms. Curr. Op. Chem. Bio., 2002, 6, 619-629. (b) Gloster, T. M.; Davies, G. J. Glycosidase inhibition: assessing mimicry of the transition state. Org. Biomol. Chem., 2010, 8, 305-320. Afarinkia K.; Mahmood, F. A novel and concise synthesis of (±) 2epi-Validamine. Tetrahedron, 1999, 55, 3129-3140. (a) Afarinkia, K.; Haji Abdullahi, M.; Scowen I. J. A new, general method for the synthesis of carbasugar-sugar pseudodisacchrides. Org. Lett., 2009, 11, 5182-5184. (b) Afarinkia, K.; Haji Abdullahi, M.; Scowen I. J. A synthesis of carbasugar-sugar pseudodisaccharides via a cycloaddition-cycloreversion reaction of 2(H)-pyran-2ones. Org. Lett., 2010, 12, 5564-5566. (a) Afarinkia, K.; Nelson, T. D.; Vinader M. V.; Posner, G. H. Diels-Alder cycloadditions of 2-pyrones and 2-pyridones. Tetrahedron, 1992, 48, 9111-9171. (b) Afarinkia, K.; Daly, N. T.; GomezFarnos S.; Joshi, S. Unusual stereoselectivity in the cycloaddition of 5-bromopyrone Tetrahedron Lett., 1997, 38, 2369-2372. (c) Afarinkia, K.; Bearpark, M.; Ndibwami, A. A computational and experimental investigation of the Diels-Alder cycloadditions of halogen substituted 2(H)-pyran-2-ones. J. Org. Chem., 2005, 70, 1122-1133. For a selection see: Posner, G. H.; Dai H. Y.; Bull D. S.; Lee J. K.; Eydoux F.; Ishihara Y.; Welsh W.; Pryor N.; Petr S. Lewis acidpromoted, stereocontrolled, gram scale, Diels-Alder cycloadditions of electronically matched 2-pyrones and vinyl ethers: The critical importance of molecular sieves and the temperature of titanium coordination with the pyrone. J. Org. Chem., 1996, 61, 671-676. (b) Markó, I. E.; Evans, G. R. Efficient lanthanide catalysis in the Diels-Alder cycloaddition of electron-deficient 2-pyrones. Synlett, 1994, 431-433. (c) Juranovic A.; Kranjc, K.; Perdih, F.; Polanc, S.; Kocevar, M. Comparison of the reaction pathways and intermediate products of a microwave-assisted and high-pressure-promoted cycloaddition of vinyl-moiety-containing dienophiles on 2H-pyran-2ones. Tetrahedron, 2011, 67, 3490-3500. (d) Cho H. K.; Tam N. T.; Cho C. G. Total synthesis of (±)-aspidospermidine starting from 3ethyl-5-bromo-2-pyrone. Bull. Korean Chem. Soc., 2010, 31, 33823384. Posner, G. H.; Afarinkia, K.; Dai, H. An improved synthesis of 3bromo-2(H)-pyranone. Org. Synth., 1995, 73, 231-239. For other examples of introducing substitution at the 5-position of 2-pyrones see: (a) Afarinkia K.; Berna-Canovas, J. Diels-Alder cycloaddition of 5-aryl-2-pyrones. Tetrahedron Lett., 2000, 41, 49554959. (b) Liu, Z.; Meinwald, J. 5-(Trimethylstannyl)-2H-pyran-2one and 3-(trimethylstannyl)-2H-pyran-2-one: New 2H-pyran-2-
Received: March 21, 2013
Revised: June 10, 2013
Accepted: July 05, 2013
[11]
[12] [13] [14] [15]
[16] [17]
[18]
[19]
[20]
[21]
[22] [23]
[24]
Current Medicinal Chemistry, 2013, Vol. 20, No. 30 3801 one synthons. J. Org. Chem., 1996, 61, 6693-6699. (c) Gravett, E. C.; Hilton, P.J.; Jones, K.; Peron, J. M. A new route to 5-aryl and 5-heteroaryl-2-pyrones via Suzuki coupling of a 2-pyrone-5boronate ester. Synlett, 2003, 253-255. Corey, E. J.; Bass, J. D.; LeMathieu, R.; Mitra R. B. A study of the photochemical reactions of 2-cyclohexenones with substituted olefins. J. Am. Chem. Soc., 1964, 86, 5570-5583. Tomisawa, H.; Fujita, R.; Noguchi, K. J.; Hongo, H. Studies on 1alkyl-2(1H)-pyridone derivatives. Chem. Pharm. Bull., 1970, 18, 941-946. Afarinkia, K.; Ndibwami, A. A general synthesis of phenantridinone alkaloids. Synlett, 2007, 1940-1944. Rose, D. R. Structure, mechanism and inhibition of Golgi alphamannosidase II. Curr. Op. Struct. Biol., 2012, 22, 558-562. Dennis, J. W.; Granovsky, M.; Warren C. E. Glycoprotein glycosidation and cancer progression. Biochim. Biophys. Acta, 1999, 1473, 21-34. (b) Dube H. D.; Bertozzi C. R. Glycans in cancer and inflammation: potential for therapeutics and diagnostics. Nat. Rev. Drug Disc., 2005, 4, 477-488. Ramana, C. V.; Vasella, A. Sugar-derived di- and tetrahydropyridazinones: Synthesis of new glycosidase inhibitors. Helv. Chim. Acta, 2000, 83, 1599-1610. (a) You, N.; Liu, W. H.; Wang, T.; Ji, R.; Wang, X.; Gong, Z. B.; Dou, K. F.; Tao, K. S. Swainsonine inhibits growth and potentiates the cytotoxic effect of paclitaxel in hepatocellular carcinoma in vitro and in vivo. Oncol. Rep., 2012, 28, 2091-2100. (b) Li, Z. C.; Xu, X. G.; Huang, Y.; Ding, L.; Wang, Z. S.; Yu, G. S.; Xu, D.; Li, W.; Tong, D. W. Swainsonine activates mitochondria-mediated apoptotic pathway in human lung cancer A549 cells and retards the growth of lung cancer xenografts. Int. J. Biol. Sci., 2012, 8, 394405. Englebienne P.; Fiaux H.; Kuntz D. A.; Corbeil C. R.; Gerber-Lemaire S.; Rose D. R.; Moitessier N. Evaluation of docking programs for predicting binding of Golgi alpha-mannosidase II inhibitors: A comparison with crystallography. Proteins, 2007, 69, 160-176. Kavelekar, L. M.; Kuntz, D. A.; Xin, W.; Johnston, B. D.; Svensson, B.; Rose, D. R.; Pinto, B. M. 5-Thio-D-glycopyranosylamines and their amidinium salts as potential transition-state mimics of glycosyl hydrolases: synthesis, enzyme inhibitory activities, X-ray crystallography, and molecular modeling. Tetrahedron Asymm., 2005, 16, 1035-1046. Shah, N.; Kuntz, D. A.; Rose, D. R. Comparison of kifunensine and 1-deoxymannojirimycin binding to class I and II alphamannosidases demonstrates different saccharide distortions in inverting and retaining catalytic mechanisms. Biochemistry, 2003, 42, 13812-13816. van den Elsen, J. M.; Kuntz, D. A.; Rose, D. R. Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells. EMBO J., 2001, 20, 3008-3017. Molecular Operating Environment (MOE), 2012.10; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2012. Howard, S.; He, S.; Withers, S. G. Identification of the active site nucleophile in jack bean alpha-mannosidase using 5-fluoro-beta-Lguloxyl fluoride. J. Biol. Chem., 1998, 273, 2067-2072. Kuntz D. A.; Tarling C. A.; Withers S. G.; Rose D. R. Structural analysis of Golgi alpha-mannosidase II inhibitors identified from a focused glycosidase inhibitor screen. Biochemistry, 2008, 47, 10058-10068.
