Skip to main content
SpringerLink
Log in

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

  • Protocol
  • First Online:
Drug Design and Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 716))

Abstract

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening ­studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

δ-OR:

Delta-opioid receptor

Ado:

8-Amino-3,6-dioxaoctanoyl

Arsenazo (III):

2,7-Bis(2-arsenophenylazo)-1,8-dihydroxyaphthalene-3,6-disulfonic acid

BB:

Bromophenol blue

Boc:

tert-Butyloxycarbonyl

CCK(6):

Nle-Gly-Trp-Nle-Asp-Phe-NH2

CCK(8):

Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2

CCK2R:

Cholecystokinin receptor subtype 2

CDI:

N,N′-carbonyldiimidazole

CEST:

Chemical exchange saturation transfer

CH3CN:

Acetonitrile

CT:

Computed tomography

Cy5:

Cyanine 5 dye

DCM:

Dichloromethane

DELFIA:

Dissociation-enhanced lanthanide fluoroimmunoassay

DIC:

N,N′-diisopropylcarbodiimide

DIEA:

Diisopropylethylamine

DMBA:

1,3-Dimethylbarbituric acid

DMEM:

Dulbecco’s modified eagle medium

DMF:

N,N′-dimethylformamide

DMSO:

Dimethylsulfoxide

Dmt:

2′,6′-Dimethyl-l-tyrosine

DOTA:

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid

DPLCE:

c[dPen2,Cys5]enkephalin

DTPA:

Diethylenetriamine-N,N,N′,N′,N²-pentaacetic acid

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

EDT:

1,2-Ethanedithiol

ESI-MS:

Electrospray ionization-mass spectrometry

Fmoc:

(9H-fluoren-9-ylmethoxy)carbonyl

FT-ICR:

Fourier transform-ion cyclotron resonance

HBTU:

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate

hMC4R:

Human melanocortin-4 receptor

HOBt:

N-hydroxybenzotriazole

HOCt:

6-Chloro-1H-hydroxybenzotriazole

htBVLs:

Heterobivalent ligands

htMVL:

Heteromultivalent ligand

MALDI-TOF:

Matrix-assisted laser desorption ionization-time of flight

MRI:

Magnetic resonance imaging

MSH:

Melanocyte-stimulating hormone

MSH-7:

Ser-Nle-Glu-His-dPhe-Arg-Trp

Mtt:

4-Methyltrityl

NDP-α-MSH:

Ac-Ser-Tyr-Ser-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2

NHS:

N-hydroxysuccinimide ester

NIR:

Near-infrared

PARACEST:

Paramagnetic chemical exchange saturation transfer

Pbf:

2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl

PEG:

Polyethyleneglycol

Pego:

19-Amino-5-oxo-3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid

RP-HPLC:

Reverse-phase high-performance liquid chromatography

SPECT:

Single photon emission computed tomography

SPPS:

Solid-phase peptide synthesis

TA:

Thioanisole

tBu:

tert-butyl

TFA:

Trifluoroacetic acid

THF:

Tetrahydrofuran

Tic:

1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid

TIS:

Triisopropylsilane

TRL:

Time-resolved luminescence

Trt:

Triphenylmethyl (trityl)

References

  1. Josan, J. S., Vagner, J., Handl, H. L., Sankaranarayanan, R., Gillies, R. J., and Hruby, V. J. (2008) Solid-phase synthesis of heterobivalent ligands targeted to melanocortin and cholecystokinin receptors. Int J Pep Res Ther. 14: 293–300.

    Article  CAS  Google Scholar 

  2. Vagner, J., Xu, L., Handl, H., Josan, J. S., Morse, D. L., Mash, E. A., Gillies, R. J., and Hruby, V. J. (2008) Heterobivalent ligands crosslink multiple cell-surface receptors: the human melanocortin-4 and delta-opioid receptors. Angew Chem Int Ed. 47: 1685–1688.

    Article  CAS  Google Scholar 

  3. Xu, L., Vagner, J., Josan, J., Lynch, R. M., Morse, D. L., Baggett, B., Han, H., Mash, E. A., Hruby, V. J., and Gillies, R. J. (2009) Enhanced targeting with heterobivalent ligands. Mol Cancer Ther. 8: 2356–2365.

    Article  PubMed  CAS  Google Scholar 

  4. Sokolov, K., Follen, M., and Richards-Kortum, R. (2002) Optical spectroscopy for detection of neoplasia. Curr Opin Chem Biol. 6: 651–658.

    Article  PubMed  CAS  Google Scholar 

  5. Ballou, B., Ernst, L. A., and Waggoner, A. S. (2005) Fluorescence imaging of tumors in vivo. Curr Med Chem. 12: 795–805.

    Article  PubMed  CAS  Google Scholar 

  6. Frangioni, J. V. (2003) In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol. 7: 626–634.

    Article  PubMed  CAS  Google Scholar 

  7. Sevick-Muraca, E. M., Houston, J. P., and Gurfinkel, M. (2002) Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. Curr Opin Chem Biol. 6: 642–650.

    Article  PubMed  CAS  Google Scholar 

  8. Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem. 4: 105–111.

    Article  PubMed  CAS  Google Scholar 

  9. Handl, H. L., Vagner, J., Yamamura, H., Hruby, V. J., and Gillies, R. J. (2004) Lanthanide-based time-resolved fluorescence of in cyto ligand–receptor interactions. Anal Biochem. 330: 242–250.

    Article  PubMed  CAS  Google Scholar 

  10. Handl, H. L., and Gillies, R. J. (2005) Lanthanide-based luminescent assays for ligand-receptor interactions. Life Sci. 77: 361–371.

    Article  PubMed  CAS  Google Scholar 

  11. Pandya, S., Yu, J., and Parker, D. (2006) Engineering emissive europium and terbium complexes for molecular imaging and sensing. Dalton Trans. 23: 2757–2766.

    Article  PubMed  Google Scholar 

  12. Selvin, P. (2002) Principles and biophysical applications of lanthanide-based probes. Annu Rev Biophys Biomol Struct. 31: 275–302.

    Article  PubMed  CAS  Google Scholar 

  13. Steinkamp, T., and Karst, U. (2004) Detection strategies for bioassays based on luminescent lanthanide complexes and signal amplification. Anal Bioanal Chem. 380: 24–30.

    Article  PubMed  CAS  Google Scholar 

  14. Parker, D. (2004) Excitement in f block: structure, dynamics and function of nine-coordinate chiral lanthanide complexes in aqueous media. Chem Soc Rev. 33: 156–165.

    Article  PubMed  CAS  Google Scholar 

  15. Thunus, L., and Lejeune, R. (1999) Overview of transition metal and lanthanide complexes as diagnostic tools. Coord Chem Rev. 184: 125–155.

    Article  Google Scholar 

  16. Josan, J. S., Morse, D. L., Xu, L., Trissal, M., Baggett, B., Davis, P., Vagner, J., Gillies, R. J., and Hruby, V. J. (2009) Solid-phase synthetic strategy and bioevaluation of a Labeled δ-opioid receptor ligand Dmt-Tic-Lys for in vivo imaging. Org Lett. 11: 2479–2482.

    Article  PubMed  Google Scholar 

  17. Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J., and Gillies, R. J. (2005) Development of a lanthanide-based assay for detection of receptor–ligand interactions at the δ-opioid receptor. Anal Biochem. 343: 299–307.

    Article  PubMed  CAS  Google Scholar 

  18. De-Silva, C. R., Vagner, J., Lynch, R., Gillies, R. J., and Hruby, V. J. (2010) Optimization of time-resolved fluorescence assay for detection of Eu-DOTA labeled ligand-receptor interactions. Anal Biochem. 398: 15–23.

    Article  PubMed  CAS  Google Scholar 

  19. Leon-Rodriguez, L. M. D., and Kovacs, Z. (2008) The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 19: 391–402.

    Article  PubMed  Google Scholar 

  20. Uusijärvi, H., Bernhardt, P., Rösch, F., Maecke, H. R., and Forssell-Aronsson, E. (2006) Electron- and positron-emitting radiolanthanides for therapy: aspects of dosimetry and production. J Nucl Med. 47: 807–814.

    PubMed  Google Scholar 

  21. Merbach, A. F., and Toth, E. (2001) The chemistry of contrast agents in medical magnetic resonance imaging. Wiley, New York.

    Google Scholar 

  22. Zhang, S., Merritt, M., Woessner, D. E., Lenkinski, R. E., and Sherry, A. D. (2003) PARACEST agents: modulating MRI ­contrast via water proton exchange. Acc Chem Res. 36: 783–790.

    Article  PubMed  CAS  Google Scholar 

  23. Aime, S., Barge, A., Delli Castelli, D., Fedeli, F., Mortillaro, A., Nielsen, F. U., and Terreno, E. (2002) Paramagnetic lanthanide (III) complexes as pH sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications. Magn Reson Med. 47: 639–648.

    Article  PubMed  CAS  Google Scholar 

  24. Yoo, B., Sheth, V., and Pagel, M. D. (2009) An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis. Tet Lett. 50: 4459–4462.

    Article  CAS  Google Scholar 

  25. Ali, M. M., Liu, G., Shah, T., Flask, C. A., and Pagel, M. D. (2009) Using two chemical exchange saturation transfer magnetic resonance imaging contrast agents for molecular imaging studies. Acc Chem Res. 42: 915–924.

    Article  PubMed  CAS  Google Scholar 

  26. Yoo, B., and Pagel, M. D. (2008) An overview of responsive MRI contrast agents for molecular imaging. Front Biosci. 13: 1733–1752.

    Article  PubMed  CAS  Google Scholar 

  27. Yoo, B., and Pagel, M. D. (2006) A PARACEST MRI contrast agent to detect enzyme activity. J Am Chem Soc. 128: 14032–14033.

    Article  PubMed  CAS  Google Scholar 

  28. Yoo, B., Raam, M., Rosenblum, R., and Pagel, M. D. (2007) Enzyme-responsive PARACEST MRI contrast agents: a new biomedical imaging approach for studies of the proteasome. Contrast Media Mol Imaging. 2: 189–198.

    Article  PubMed  CAS  Google Scholar 

  29. Grieco, P., Lavecchia, A., Cai, M., Trivedi, D., Weinberg, D., MacNeil, T., Van der Ploeg, L. H., and Hruby, V. J. (2002) Structure-activity studies of the melanocortin peptides: discovery of potent and selective affinity antagonists for the hMC3 and hMC4 receptors. J Med Chem. 45: 5287–5294.

    Article  PubMed  CAS  Google Scholar 

  30. Martinez-Zaguilan, R., Tompkins, L. S., Gillies, R. J., and Lynch, R. M. (1999) Simultaneous analysis of intracellular pH and Ca2+ from cell populations. Meth Mol Biol. 114: 287–306.

    Google Scholar 

  31. Edwards, W. B., Fields, C. G., Anderson, C. J., Pajeau, T. S., Welch, M. J., and Fields, G. B. (1994) Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs. J Med Chem. 37: 3749–3757.

    Article  PubMed  CAS  Google Scholar 

  32. Krchnák, V., Vágner, J., and Lebl, M. (1988) Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator. Int J Pept Prot Res. 32: 415–416.

    Article  Google Scholar 

  33. Bräse, S., Kirchhoff, J. H., and Köbberling, J. (2003) Palladium-catalysed reactions in solid phase organic synthesis. Tetrahedron. 59: 885–939.

    Article  Google Scholar 

  34. Gomez-Martinez, P., Dessolin, M., Guibé, F., and Albericio, F. (1999) Nα-Alloc temporary protection in solid-phase peptide synthesis. The use of amine–borane complexes as allyl group scavengers. J Chem Soc Perkin Trans. 1: 2871–2874.

    Article  Google Scholar 

  35. Williams, R. M., Aldous, D. J., and Aldous, S. C. (1990) General synthesis of β, γ-alkynylglycine derivatives. J Org Chem. 55: 4657–4663.

    Article  CAS  Google Scholar 

  36. Moore, D. A. (2008) Selective trialkylation of cyclen with tert-butyl bromoacetate. Org Synth. 85: 10–14.

    CAS  Google Scholar 

  37. Ali, M., Yoo, B., and Pagel, M. D. (2009) Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MRI. Mol Pharm. 6: 1409–1416.

    Article  PubMed  CAS  Google Scholar 

  38. Liu, G., Ali, M. M., Yoo, B., Griswold, M. A., Tkach, J. A., and Pagel, M. D. (2009) PARACEST MRI with improved temporal resolution. Magn Reson Med. 61: 399–408.

    Article  PubMed  Google Scholar 

  39. Liu, G., Li, Y., and Pagel, M. D. (2007) Design and characterization of a new irreversible responsive PARACEST MRI contrast agent that detects nitric oxide. Magn Reson Med. 58: 1249–1256.

    Article  PubMed  CAS  Google Scholar 

  40. Josan, J. S. (2008) Heteromultivalent ligands directed targeting of cell-surface receptors – implications in cancer diagnostics & therapeutics. Ph.D. dissertation. The University of Arizona, Tucson.

    Google Scholar 

  41. Dakubu, S. (1992) Method of determining a biological substance involving labelling with a metal chelate. US Patent 5,124,268.

    Google Scholar 

  42. Wilkinson, D. (1999) A one-step fluorescent detection method for lipid fingerprints; Eu(TTA)3.2TOPO. Forensic Sci Int. 99: 5–23.

    Article  PubMed  CAS  Google Scholar 

  43. Albericio, F., Annis, I., Royo, M., and Barany, G. (2000) Preparation and handling of peptides containing methionine and cysteine. In: Chan, W. C., White P. D. (eds) Fmoc solid phase peptide synthesis, 1st ed., Oxford University Press, Oxford, pp 77–114.

    Google Scholar 

  44. Annis, I., Hargittai, B., and Barany, G. (1997) Disulfide bond formation in peptides. Meth Enzymol. 289: 198–221.

    Article  PubMed  CAS  Google Scholar 

  45. Chen, L., Annis, I., and Barany, G. (2001) Disulfide bond formation in peptides. Curr Protocols Prot Sci. 18.16.11–18.16.19.

    Google Scholar 

  46. Fields, G. B., Lauer-Fields, J. L., Liu, R., and Barany, G. (1992) Principles and practice of solid phase peptide synthesis. In: Grant, G. A. (ed) Synthetic Peptides: A User’s Guide, 2nd ed., Oxford University Press, Oxford, pp 93–219.

    Google Scholar 

  47. Gisin, B. F. (1972) The monitoring of reactions in solid-phase peptide synthesis with picric acid. Anal Chim Acta. 58: 248–249.

    Article  PubMed  CAS  Google Scholar 

  48. Rowatt, E., and Williams, R. J. (1989) The interaction of cations with the dye Arsenazo III. Biochem J. 259: 295–298.

    PubMed  CAS  Google Scholar 

  49. Barge, A., Cravotto, G., Gianolio, E., and Fedeli, F. (2006) How to determine free Gd and free ligand in solution of Gd chelates. A technical note. Contrast Media Mol Imaging. 1: 184–188.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Prof. Robert J. Gillies and his team for development of various cell lines and animal model for δOR tumor animal model described in this work. This work was supported by the National Cancer Institute through NIH Grant R21CA133455-01, R01 CA09736, and R01 CA 123547, and by the U.S. Army Medical Research and Materiel Command under W81XWH-04-1-0731. This work was also supported by the Northeastern Ohio Animal Imaging Resource Center, an NIH-funded program, R24CA110943.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Arizona, Tucson, AZ, USA

    Jatinder S. Josan, Channa R. De Silva, Byunghee Yoo, Mark D. Pagel & Victor J. Hruby

  2. Bio5 Institute, University of Arizona, Tucson, AZ, USA

    Ronald M. Lynch & Josef Vagner

Authors
  1. Jatinder S. Josan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Channa R. De Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Byunghee Yoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ronald M. Lynch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark D. Pagel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Josef Vagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Victor J. Hruby
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Victor J. Hruby .

Editor information

Editors and Affiliations

  1. College of Pharmacy, Dept. Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Bienville Drive 1800, Monroe, 71201, Louisiana, USA

    Seetharama D. Satyanarayanajois

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Josan, J.S. et al. (2011). Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging. In: Satyanarayanajois, S. (eds) Drug Design and Discovery. Methods in Molecular Biology, vol 716. Humana Press. https://doi.org/10.1007/978-1-61779-012-6_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-012-6_6

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-011-9

  • Online ISBN: 978-1-61779-012-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation