Glycans can be imaged by metabolic labeling with azidosugars followed by chemical reaction with imaging probes; however, tissue-specific labeling is difficult to achieve. Here we describe a strategy for the use of a caged metabolic precursor that is activated for cellular metabolism by enzymatic cleavage. An N-azidoacetylmannosamine derivative caged with a peptide substrate for the prostate-specific antigen (PSA) protease was converted to cell-surface azido sialic acids in a PSA-dependent manner. The approach has applications in tissue-selective imaging of glycans for clinical and basic research purposes. © 2010 American Chemical Society

Baskin J. M.

Blum G.

Carolyn R. Bertozzi

Chang P. V.

Danielle H. Dube

de Groot F. M.

Denmeade S. R.

Ellen M. Sletten

Jacobs C. L.

Jiang T.

Jones G. B.

Khan S. R.

Massoud T. F.

Pamela V. Chang

Prescher J. A.

Sletten E. M.

Varki A.

Weissleder R.

Zhang S.

English

PubMed

Crossref

Journal of the American Chemical Society

A Strategy for the Selective Imaging of Glycans Using Caged Metabolic Precursors

Chang, Pamela V.

Dube, Danielle H.

Sletten, Ellen M.

Bertozzi, Carolyn R.

Bowdoin College

Bowdoin College Bowdoin Digital Commons Chemistry Faculty Publications Faculty Scholarship and Creative Work 7-21-2010 A strategy for the selective imaging of glycans using caged metabolic precursors Pamela V. Chang University of California, Berkeley Danielle H. Dube Lawrence Berkeley National Laboratory Ellen M. Sletten University of California, Berkeley Carolyn R. Bertozzi University of California, Berkeley Follow this and additional works at: https://digitalcommons.bowdoin.edu/chemistry-faculty-publications Recommended Citation Chang, Pamela V.; Dube, Danielle H.; Sletten, Ellen M.; and Bertozzi, Carolyn R., "A strategy for the selective imaging of glycans using caged metabolic precursors" (2010). Chemistry Faculty Publications. 12. https://digitalcommons.bowdoin.edu/chemistry-faculty-publications/12 This Article is brought to you for free and open access by the Faculty Scholarship and Creative Work at Bowdoin Digital Commons. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of Bowdoin Digital Commons. For more information, please contact mdoyle@bowdoin.edu, a.sauer@bowdoin.edu. A Strategy for the Selective Imaging of Glycans Using Caged MetabolicPrecursorsPamela V. Chang, Danielle H. Dube,† Ellen M. Sletten, and Carolyn R. Bertozzi*Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, UniVersity ofCalifornia, Berkeley, California 94720, and The Molecular Foundry, Lawrence Berkeley National Laboratory,Berkeley, California 94720Received February 12, 2010; E-mail: crb@berkeley.eduAbstract: Glycans can be imaged by metabolic labeling withazidosugars followed by chemical reaction with imaging probes;however, tissue-specific labeling is difficult to achieve. Here wedescribe a strategy for the use of a caged metabolic precursorthat is activated for cellular metabolism by enzymatic cleavage.An N-azidoacetylmannosamine derivative caged with a peptidesubstrate for the prostate-specific antigen (PSA) protease wasconverted to cell-surface azido sialic acids in a PSA-dependentmanner. The approach has applications in tissue-selective imag-ing of glycans for clinical and basic research purposes.A fundamental challenge in molecular imaging is the design ofreagents that report on cell- or tissue-specific molecular processes. Apopular approach to achieving such selectivity is the design of cagedprobes that are activated by enzymes produced by the target cells, aprototypical example being cancer-associated proteases.1 Alternatively,probes can be conjugated to targeting elements that bind, noncovalentlyor covalently, to cell-surface markers.2 We have been exploring thislatter approach in the context of imaging cell-surface glycans. Thesebiopolymers, collectively termed the glycome, change in structure andexpression during development and cancer progression as well as manyother physiological processes.3 We previously reported that broadsectors of the glycome can be imaged in model organisms using atwo-step procedure: (1) metabolic labeling of the glycans withazidosugar precursors and (2) chemical reaction of azide-labeled cell-surface glycans in ViVo via Cu-free click chemistry with difluorinatedcyclooctyne (DIFO) probes.4The tissue selectivity of this approach is limited by the breadth ofglycans targeted by a single metabolic precursor. For example,treatment of mice with peracetylated N-azidoacetylmannosamine(Ac4ManNAz) leads to widespread labeling of glycans with N-azidoacetyl sialic acid (SiaNAz) in numerous tissues (e.g., liver, heart,kidney, and intestines) as well as serum glycoproteins.5 The broaddistribution of the azidosugar can undermine experiments that seek toprobe glycomic changes in a single organ as a function of time ordisease. One means to achieve tissue selectivity in glycan imaging isto restrict azidosugar metabolism to the cells of interest. Tissue-specificenzyme activities such as those mentioned above might be exploitedto achieve this goal. Here, we describe a strategy for cell-selectiveglycan imaging using a caged metabolic precursor that is activated bya protease (Figure 1A).We synthesized a variant of Ac4ManNAz in which the 6-hydroxygroup was conjugated through a self-immolating linker to a peptidesubstrate for the prostate-specific antigen (PSA) protease (1, Figure1B). PSA is a serine protease that is secreted at low levels by normalprostatic glandular cells but is highly upregulated by prostate cancercells.6 The enzyme has been widely targeted with caged drugs andimaging reagents in cell culture and in animal models.6 The hexapep-tide Mu-HSSKLY (Mu ) morpholino ureidyl) was chosen as a PSAsubstrate based on reports of its high selectivity for this enzyme overother ubiquitous serine proteases.6c,7 Upon cleavage of the peptide,the p-aminobenzyl alcohol (PABA) linker in 1 spontaneously fragmentsto release 1,3,4-tri-O-acetyl-N-azidoacetylmannosamine (Ac3ManNAz),carbon dioxide, and an iminoquinone methide intermediate that issubsequently quenched by water (Figure 1B).8 This process is knownto occur rapidly and should therefore limit the diffusion of the releasedmetabolic substrate from its target cell (for discussion see Figure S1legend). Compound 1 was synthesized as a mixture of sugar anomersanalogously to the route developed by Jones et al. (Scheme S1).6cGiven the importance of hydroxy group acylation for the cellular uptakeof Ac4ManNAz,9 we verified that Chinese hamster ovary (CHO) andprostate cancer (PC-3) cells incubated with Ac3ManNAz could produceSiaNAz residues in their cell-surface glycans (Figure S1).To confirm that 1 can serve as a substrate for PSA, the compoundwas incubated in Vitro with active enzyme or, as negative controls,buffer only or heat-killed (HK) PSA. These enzymatic reactions wereanalyzed by reversed-phase HPLC and mass spectrometry. Incubation† Current address: Department of Chemistry, Bowdoin College, Brunswick, ME04011.Figure 1. Strategy for tissue-specific release of Ac3ManNAz via enzymaticactivation. (A) (i) A nonmetabolizable caged azidosugar serves as a substratefor a secreted, cancer-specific protease, releasing Ac3ManNAz. (ii) Thisazidosugar is then metabolized by the cell and incorporated into cell-surfaceglycans. (iii) The azide-labeled glycans are detected via Cu-free click chemistryusing DIFO reagents. (B) Caged azidosugar used in this study (1). Cleavageof the indicated bond (dashed line) by the prostate-specific antigen (PSA)protease results in the release of a linker, the peptide, and Ac3ManNAz.Published on Web 06/22/201010.1021/ja101080y  2010 American Chemical Society9516 9 J. AM. CHEM. SOC. 2010, 132, 9516–9518Downloaded via BOWDOIN COLG on May 17, 2023 at 19:36:53 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.of 1 with PSA resulted in the release of Mu-HSSKLY as the majorpeptide product, along with Ac3ManNAz (Figure 2A). Minor productswere also observed. These included Mu-HSSKLY-PABA, presumablyproduced by nonenzymatic carbonate hydrolysis, as well as mono-deacetylated 1 and products formed by migration of acetyl groupswithin 1. Control reactions lacking PSA (Figure 2B) or with HK PSA(Figure 2C) produced only nonenzymatic hydrolysis and acetylmigration products.We next tested the performance of 1 as a caged substrate formetabolic glycan labeling. CHO cells were incubated with 1 at variousconcentrations (0-100 µM) in the presence of PSA, no enzyme, orHK PSA for 12 h at 37 °C. The cells were then washed and labeledwith a DIFO-biotin conjugate,4b incubated with fluorescein isothio-cyanate-labeled avidin (FITC-avidin), and analyzed by flow cytometry.We observed labeling that was both PSA- and substrate concentration-dependent, suggesting that the signal is due to enzymatic activationof 1 (Figure 3A). In a separate experiment, we demonstrated that thelabeling intensity correlates with PSA concentration (Figure S2).Additionally, we verified that treatment of PC-3 cells with 1 resultedin PSA-dependent metabolic labeling (Figure S3). In the absence ofPSA or with HK PSA, both CHO and PC-3 cells exhibited modestbackground labeling that likely reflects low levels of Ac3ManNAzproduced by nonenzymatic carbonate hydrolysis (Figures 3B and S3).Importantly, we verified that 1 did not cause any cytotoxicity byincubating CHO cells labeled as above with phycoerythrin-conjugatedannexin V, a marker of apoptosis (Figure S4).Finally, we tested 1 as an enzyme-activatable metabolic substratefor glycan imaging. CHO cells were incubated with 1 in the presenceof PSA or HK PSA for 12 h at 37 °C. The cells were then washedand labeled with DIFO-biotin, followed by quantum-dot-conjugatedstreptavidin. We observed substantial cell-surface labeling of cellstreated with 1 and PSA (Figure 4A) and minimal fluorescence on cellstreated with 1 and HK PSA (Figure 4B).In conclusion, we have developed a strategy for targeted metabolismof azidosugars using an enzymatically activated substrate. While wechose PSA to demonstrate proof-of-concept, it should be noted thatthe concentrations of PSA employed in our studies are physiologicallyrelevant; i.e., they are similar to the levels of PSA secreted by bothprostate cancer xenografts in mice and prostate tumor tissue obtainedfrom human patients.10 In addition, many cancers, including prostatecancer, are known to express elevated levels of sialic acid comparedto surrounding tissue.11 Thus, clinical imaging applications may beworth pursuing. More generally, however, the approach has promisefor use in tissue-specific glycan imaging, a major future direction.Acknowledgment. This work was supported by NIH GrantGM058867. We thank A. Lo for technical assistance and J. Baskinfor critical reading of the manuscript. P.V.C. and D.H.D. weresupported by NSF predoctoral fellowships. P.V.C. was also supportedby an ACS Division of Medicinal Chemistry predoctoral fellowship.E.M.S. was supported by an ACS Division of Organic Chemistrypredoctoral fellowship.Supporting Information Available: Synthetic procedures and ad-ditional data. This material is available free of charge via the Internet athttp://pubs.acs.org.References(1) (a) Blum, G.; von Degenfeld, G.; Merchant, M. J.; Blau, H. M.; Bogyo,M. Nat. Chem. Biol. 2007, 3, 668–677. (b) Jiang, T.; Olson, E. S.; Nguyen,Q. T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.2004, 101, 17867–17872. (c) Weissleder, R.; Tung, C. H.; Mahmood, U.;Bogdanov, A., Jr. Nat. Biotechnol. 1999, 17, 375–378.(2) Massoud, T. F.; Gambhir, S. S. Genes DeV. 2003, 17, 545–580.(3) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.;Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, 2nded.; Cold Spring Harbor Laboratory Press: New York, 2008.(4) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–6998. (b) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc.Natl. Acad. Sci. U.S.A. 2007, 104, 16793–16797.(5) (a) Chang, P. V.; Prescher, J. A.; Sletten, E. M.; Baskin, J. M.; Miller,I. A.; Agard, N. J.; Lo, A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A.2010, 107, 1821–1826. (b) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R.Nature 2004, 430, 873–877.(6) (a) Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A. V.; Isaacs,J. T. Cancer Res. 1998, 58, 2537–2540. (b) Khan, S. R.; Denmeade, S. R.Prostate 2000, 45, 80–83. (c) Jones, G. B.; Crasto, C. F.; Mathews, J. E.;Figure 2. Compound 1 is a substrate for PSA in Vitro. Shown are HPLCtraces of in Vitro 6 h enzymatic reactions of 1 (500 µM) in 50 mM Tris, 0.1 MNaCl, pH 7.8, with (A) active PSA (50 µg/mL), (B) buffer only, or (C) HKPSA (50 µg/mL). The identities of the various species based on massspectrometry are indicated on the traces. *Monoacetylated Mu-HSSKLY-PABA, **Monodeacetylated 1, and ***Isomers of 1.Figure 3. Cell-selective metabolic labeling of glycans using 1 and PSA. Flowcytometry analysis of CHO cells treated with (A) various concentrations of 1(0-100 µM) and PSA (50 µg/mL, squares) or buffer only (circles) or (B) 1(100 µM) and either buffer only (-), HK PSA (50 µg/mL, HK), or PSA (50µg/mL, +). Cells were then labeled with DIFO-biotin (100 µM) and FITC-avidin. Error bars represent the standard deviation from the mean of threereplicate samples. MFI ) mean fluorescence intensity in arbitrary units (AU).Figure 4. Selective imaging of cells using 1 in the presence of PSA.Fluorescence microscopy analysis of CHO cells treated with 1 (100 µM) and(A) PSA (50 µg/mL) or (B) HK PSA (50 µg/mL), followed by DIFO-biotin(100 µM) and a quantum dot 605-streptavidin conjugate. Green ) Texas Redchannel; Blue ) DAPI channel. Scale bar ) 20 µm.J. AM. CHEM. SOC. 9 VOL. 132, NO. 28, 2010 9517C O M M U N I C A T I O N SXie, L.; Mitchell, M. O.; El-Shafey, A.; D’Amico, A. V.; Bubley, G. J.Bioorg. Med. Chem. 2006, 14, 418–425.(7) Denmeade, S. R.; Lou, W.; Lovgren, J.; Malm, J.; Lilja, H.; Isaacs, J. T.Cancer Res. 1997, 57, 4924–4930.(8) de Groot, F. M.; Damen, E. W.; Scheeren, H. W. Curr. Med. Chem. 2001,8, 1093–1122.(9) Jacobs, C. L.; Yarema, K. J.; Mahal, L. K.; Nauman, D. A.; Charters, N. W.;Bertozzi, C. R. Methods Enzymol. 2000, 327, 260–275.(10) Denmeade, S. R.; Sokoll, L. J.; Chan, D. W.; Khan, S. R.; Isaacs, J. T.Prostate 2001, 48, 1–6.(11) (a) Zhang, S.; Cordon-Cardo, C.; Zhang, H. S.; Reuter, V. E.; Adluri, S.;Hamilton, W. B.; Lloyd, K. O.; Livingston, P. O. Int. J. Cancer 1997, 73,42–49. (b) Zhang, S.; Zhang, H. S.; Cordon-Cardo, C.; Reuter, V. E.;Singhal, A. K.; Lloyd, K. O.; Livingston, P. O. Int. J. Cancer 1997, 73,50–56.JA101080Y9518 J. AM. CHEM. 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A strategy for the selective imaging of glycans using caged metabolic precursors

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A Strategy for the Selective Imaging of Glycans Using Caged Metabolic Precursors

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