Wavelength‐Selective Uncaging of Oligonucleotides

Alexandre Rodrigues‐Correia1, Patrick Seyfried1, Alexander Heckel1

1 Institute for Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe‐University Frankfurt, Frankfurt
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.11
DOI:  10.1002/0471142700.nc0611s57
Online Posting Date:  June, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Caged compounds are light‐sensitive molecules with temporarily inactivated biological function. The active compound is released upon irradiation, in which exact spatial and temporal control is accomplished. Beyond this inherently irreversible concept of triggering, the idea of multi‐wavelength uncaging provides experiments with more complexity. This unit describes the syntheses of protected nucleoside phosphoramidites of caged dTNpHP [pHP = (p‐hydroxyphenacyl)], dTDEACM {DEACM = [(7‐diethylaminocoumarin‐4‐yl)methyl]} or a dCNDBF {NDBF = [1‐(3‐nitrodibenzofuran‐1‐yl)ethyl]} modification on the nucleobase, their incorporation in oligonucleotides, characterization, and their wavelength‐selective uncaging up to four levels. Curr. Protoc. Nucleic Acid Chem. 57:6.11.1‐6.11.32. © 2014 by John Wiley & Sons, Inc.

Keywords: uncaging; photolabile‐protecting groups; selective photochemistry; quantum yields

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of N3‐Protected pHP‐Caged Thymidine Phosphoramidite
  • Basic Protocol 2: Preparation of Protected DEACM‐Caged Thymidine Phosphoramidite
  • Basic Protocol 3: Preparation of Protected NDBF‐Caged Deoxycytidine Phosphoramidite
  • Support Protocol 1: Preparation of the Caging Group Precursor Tips‐pHP‐Br
  • Support Protocol 2: Preparation of the Caging Group Precursor DEACM‐OH
  • Support Protocol 3: Preparation of the Caging Group Precursor NDBF‐NH2
  • Basic Protocol 4: Preparation of Modified Oligonucleotides
  • Support Protocol 4: Determination of Quantum Yields
  • Basic Protocol 5: Uncaging at Different Wavelengths
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of N3‐Protected pHP‐Caged Thymidine Phosphoramidite

  Materials
  • Thymidine (1)
  • Pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Ethanol (EtOH)
  • Saturated aqueous NaHCO 3
  • Distilled water
  • Anhydrous MgSO 4
  • Triethylamine (Et 3N)
  • Potassium carbonate (K 2CO 3)
  • TIPS‐pHP‐Br (20, see protocol 4)
  • Ethyl acetate
  • Cyclohexane
  • N,N‐Diisopropylethylamine (DIPEA)
  • 2‐Cyanoethoxy‐N,N‐diisopropylaminochlorophosphine
  • Acetone
  • 100‐ and 250‐mL round‐bottom flasks
  • Stir bars
  • Ice‐water bath
  • Rotary evaporator connected to a vacuum pump
  • High vacuum pump
  • Separating funnels
  • Glass frit
  • Water aspirator
  • Syringes
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of Protected DEACM‐Caged Thymidine Phosphoramidite

  Materials
  • Thymidine
  • N,N‐Dimethylformamide (DMF)
  • Imidazole
  • Tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • Cyclohexane
  • Ethyl acetate
  • Ethanol
  • Ethyl acetate
  • Distilled water
  • 1 M Hydrochlorid acid
  • Saturated aqueous NaHCO 3
  • Anhydrous MgSO 4
  • 1,2,4‐Triazole
  • Dry acetonitrile
  • Phosphoryl chloride (POCl 3)
  • Triethylamine (Et 3N)
  • Dichlomethane (CH 2Cl 2)
  • Saturated aqueous NaCl
  • DEACM‐OH (22; see protocol 5)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐en (DBU)
  • Tetrahydrofurane (THF)
  • Glacial acetic acid
  • 1 M Tetrabutylammonium fluoride in THF (TBAF)
  • Methanol
  • Dry pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 5% aqueous citric acid
  • N‐Ethyl‐N,N‐diisopropylamine (DIPEA)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • 1‐L, 250‐ and 100‐mL round‐bottom flasks
  • Stir bars
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Glass frit
  • Water aspirator
  • Ice‐water bath
  • Separating funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of Protected NDBF‐Caged Deoxycytidine Phosphoramidite

  Materials
  • 2′‐Deoxyuridine (11)
  • Imidazole
  • Tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • N,N‐Dimethylformamide (DMF)
  • Dichloromethane (CH 2Cl 2)
  • Methanol
  • Ethanol
  • Ethyl acetate
  • 1 M aqueous HCl
  • Saturated aqueous NaHCO 3
  • Saturated aqueous NaCl
  • Anhydrous MgSO 4
  • 4‐(Dimethylamino)pyridine (DMAP)
  • N,N‐Diisopropylethylamine (DIPEA)
  • 2,4,6‐Triisopropylbenzenesulfonylchloride (TIPBS‐Cl)
  • Ethyl acetate
  • Cyclohexane
  • NDBF‐NH 2 (28; see protocol 6)
  • Distilled water
  • Tetrahydrofuran (THF)
  • Tetrabutylammonium fluoride solution (TBAF) 1 M in THF
  • Pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 5% aq. Citric acid
  • Triethylamine (Et 3N)
  • 2‐Cyanoethoxy‐N,N‐diisopropylaminochlorophosphine
  • Acetone
  • 1‐L, 250‐, 100‐ and 50‐mL round‐bottom flasks
  • Stir bars
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Glass frit
  • Water aspirator
  • Ice‐water bath
  • Oil bath
  • Separating funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of the Caging Group Precursor Tips‐pHP‐Br

  Materials
  • p‐Hydroxacetophenone (18)
  • Imidazole
  • N,N‐Dimethylformamide (DMF)
  • Ethanol
  • Ethyl acetate
  • 1 M aqueous Hydrochloric acid
  • Distilled water
  • Anhydrous MgSO 4
  • Cyclohexane
  • Dichloromethane (CH 2Cl 2)
  • Copper(II) bromide (CuBr 2)
  • Chloroform
  • 250‐ and 100‐mL round bottom flasks
  • Stir bars
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Glass frit
  • Water aspirator
  • Oil bath
  • Separating funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 2: Preparation of the Caging Group Precursor DEACM‐OH

  Materials
  • 4‐Methyl‐diethylaminocoumarine (21)
  • p‐Xylene
  • Selenium dioxide (SeO 2)
  • Ethanol
  • Sodium borohydride (NaBH 4)
  • 1 M aqueous HCl
  • Distilled water
  • Saturated aqueous NaCl
  • Dichloromethane (CH 2Cl 2)
  • Anhydrous MgSO 4
  • Acetone
  • 1‐L round‐bottom flasks
  • Stir bars
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Glass frit
  • Water aspirator
  • Separating funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 3: Preparation of the Caging Group Precursor NDBF‐NH2

  Materials
  • Anhydrous AlCl 3
  • Carbon disulfide (CS 2)
  • Acetylchloride
  • Dibenzofurane (22)
  • Ice water
  • Ethyl acetate
  • 0.5 M Aqueous NaOH
  • Distilled water
  • Anhydrous MgSO 4
  • Cyclohexane
  • Zinc powder
  • Mercury (II) chloride (HgCl 2)
  • Concentrated hydrochloride acid
  • Diethyl ether
  • Glacial acetic acid
  • Fuming nitric acid
  • Methanol
  • N‐Bromosuccinimide (NBS)
  • Dibenzoylperoxide
  • Carbon tetrachloride (CCl 4)
  • Dichloromethane (CH 2Cl 2)
  • Saturated aqueous NaHCO 3
  • DMF
  • NaN 3
  • Saturated aqueous NaCl
  • Tetrahydrofurane (THF)
  • Acetonitrile
  • Triphenylphosphine (PPh 3)
  • 100‐mL, 250‐mL, 1‐L and 2‐L round‐bottom flasks
  • Stir bars
  • Oil pump
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Glass frit
  • Water aspirator
  • Separating funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Preparation of Modified Oligonucleotides

  Materials
  • DNA phosphoramidites (diluted on the synthesizer to 0.1 M in acetonitrile, using automated protocols)
  • 5′‐O‐DMTr‐N6‐(benzoyl)‐2′‐deoxyadenosine‐3′‐O‐(β‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  • 5′‐O‐DMTr‐N2‐(dimethylformamid)‐2′‐deoxguanosine‐3′‐O‐(β‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  • 5′‐O‐DMTr‐N4‐(Acetyl)‐2′‐deoxycytidine‐3′‐O‐(β‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  • 5′‐O‐DMTr‐2′‐deoxythymidine‐3′‐O‐(β‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  • Concentrated ammonium hydroxide
  • 80% (v/v) acetic acid (HOAc) in ultrapure water
  • Ultrapure water
  • DNA/RNA synthesizer
  • 2‐mL plastic reaction tubes
  • Thermo shaker
  • Centrifugal Filter (VWR, Nylon 0.45 µm, 500 µl)
  • Vacuum concentrator
  • UV spectrometer
  • ABI LV200 polystyrene columns

Support Protocol 4: Determination of Quantum Yields

  Materials
  • 1,10‐Phenanthroline
  • Aqueous H 2SO 4 (0.5 M and 0.05 M)
  • Ammonium iron (III) oxalate trihydrate [(NH 4) 2Fe(SO 4) 2]
  • Ammonium iron (II) sulfate hexahydrate [(NH 4) 3Fe(C 2O 4) 3]
  • Sodium acetate
  • Ultrapure water
  • Cuvettes (1 cm coat thickness)
  • 2‐mL reaction tubes
  • Light sources (100‐W Hg short arc lamp with a band pass filter at 313 nm and LED devices with λ max 365 nm, 440 nm and 505 nm; customized LEDs with drivers are available e.g., from Thorlabs, http://www.thorlabs.com; the pure LEDs can be obtained e.g., from Roithner, http://www.roithner‐laser.com/)
  • Spectrophotometer
  • HPLC

Basic Protocol 5: Uncaging at Different Wavelengths

  Materials
  • Modified oligonucleotides (see protocol 7)
  • Phosphate‐buffer saline (PBS; 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl), pH 7.4
  • Ultrapure water
  • 2‐mL dark reaction tubes
  • Light sources (100‐W Hg short arc lamp with a band pass filter at 313 nm and LED devices with λ max 365 nm, 440 nm, and 505 nm; customized LEDs with drivers e.g., are available e.g., from Thorlabs, http://www.thorlabs.com; the pure LEDs can be obtained e.g., from Roithner, http://www.roithner‐laser.com/)
  • HPLC
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Bochet, C.G. 2004. Chromatic orthogonality in organic synthesis. Synlett 13:2268‐2274.
  Bochet, C.G. 2006. Photochemical release of various functional groups. Pure Appl. Chem. 78:241‐247.
  Brieke, C., Rohrbach, F., Gottschalk, A., Mayer, G., and Heckel, A. 2012. Light‐controlled tools. Angew. Chem. Int. Ed. 51:8446‐8467.
  Engels, J. and Schlaeger, E.J. 1977. Synthesis, structure, and reactivity of adenosine cyclic 3′,5′‐phosphate benzyl triesters. J. Med. Chem. 20:907‐911.
  Engels, J. and Reidys, R. 1978. Synthesis and application of the photolabile guanosine 3′,5′‐phosphoric‐o‐nitrobenzylester. Cell. Mol. Life. Sci. 1:14‐15.
  Kaplan, J.H., Forbush, B. III, and Hoffman, J.F. 1978. Rapid photolatic release of adenosine 5′‐triphosphate from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17:1929‐1935.
  Klán, P., Šolomek, T., Bochet, C.G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. 2013. Photoremovable protecting groups in chemistry and biology: Reaction mechanisms and efficacy. Chem. Rev. 113:119‐191.
  Kuhn, H.J., Braslavsky, S.E., and Schmidt, R. 2004. Chemical actinometry (IUPAC technical report). Pure Appl. Chem. 76:2105‐2146.
  Menge, C. and Heckel, A. 2011. Coumarin‐Caged dG for improved wavelength‐selective uncaging of DNA. Org. Lett. 13:4620‐4623.
  Momotake, A., Lindegger, N., Niggli, E., Barsotti, R.J., and Ellis‐Davies G.C.R. 2006. The nitrodibenzofuran chromophore: A new caging group for ultra‐efficient photolysis in living cells. Nat. Methods 3:35‐40.
  Rodrigues‐Correia, A., Koeppel, M.B., Schäfer, F., Joshi, K.B., Mack, T., and Heckel, A. 2011. Comparison of the duplex‐destabilizing effects of nucleobase‐caged oligonucleotides. Anal. Bioanal. Chem. 399:441‐447.
  Rodrigues‐Correia, A., Weyel, X.M.M., and Heckel, A. 2013. Four levels of wavelength‐selective uncaging for oligonucleotides. Org. Lett. 15:5500‐5503.
  Schäfer, F., Joshi, K.B., Fichte, M.A.H., Mack, T., Wachtveitl, J., and Heckel, A. 2011. Wavelength‐selective uncaging of dA and dC residues. Org. Lett. 13:1450‐1453.
  Schönleber, R.O., Bendig, J., Hagen, V., and Giese, B. 2002. Rapid photolytic release of cytidine 50‐Diphosphate from a coumarin derivative: A new tool for the investigation of ribonucleotide reductases. Bioorg. Med. Chem. 10:97‐101.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library