Design, Synthesis, and Application of the Trimethoprim‐Based Chemical Tag for Live‐Cell Imaging

Chaoran Jing1, Virginia W. Cornish1

1 Department of Chemistry, Columbia University, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130019
Online Posting Date:  June, 2013
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Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live‐cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim‐based chemical tag (TMP‐tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity‐induced protein labeling reactions. To date, the TMP‐tag is one of the few chemical tags that enable intracellular protein labeling and high‐resolution live‐cell imaging. Here we describe the general design, chemical synthesis, and application of TMP‐tag for live‐cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP‐tags are also included. Curr. Protoc. Chem. Biol. 5:131‐155 © 2013 by John Wiley & Sons, Inc.

Keywords: chemical tag; fluorescence microscopy; live‐cell imaging; protein label

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis and Application of Noncovalent TMP‐Tag for Live‐Cell Imaging
  • Alternate Protocol 1: Synthesis and Application of Covalent TMP‐Tag for Live‐Cell Imaging
  • Alternate Protocol 2: Synthesis and Application of Fluorogenic TMP‐Tag for Live‐Cell Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Synthesis and Application of Noncovalent TMP‐Tag for Live‐Cell Imaging

  • Hydrobromic acid (HBr)
  • Trimethoprim (Sigma‐Aldrich, cat. no. T7883)
  • Sodium hydroxide (NaOH)
  • Millipore‐purified water
  • Ammonium hydroxide (28% in water, w/w)
  • Anhydrous N,N‐dimethylformamide (DMF) in sure‐seal bottle
  • Potassium carbonate (K 2CO 3)
  • Ethyl 5‐bromovalerate
  • Compressed Ar
  • Brine (saturated NaCl water solution)
  • Ethyl acetate
  • Methanol (MeOH) for chromatography
  • Dichloromethane (DCM) for chromatography
  • Silica gel
  • Deuterated methanol (CD 3OD); used to take NMR spectra
  • Hydrochloric acid (HCl)
  • Deuterated dimethyl sulfoxide (DMSO‐d6); used to take NMR spectra
  • Propane flame
  • Boc‐1‐amino‐4,7,10‐trioxa‐13‐tridecanamine (BocNH‐PEG‐NH 2), CAS: 194920‐62‐2
  • Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)
  • N,N‐Diisopropylethylamine (DIEA)
  • Trifluoroacetic acid (TFA)
  • Atto655‐NHS ester (Sigma‐Aldrich, cat. no. 76245)
  • Acetonitrile (MeCN) for HPLC
  • Dimethyl sulfoxide (DMSO)
  • Phosphate‐buffered saline (PBS; see recipe)
  • PCR primers:
  • H2B‐GFP vector (Addgene, cat. no. 11680)
  • AgeI and NotI restriction endonucleases
  • Agarose for electrophoresis
  • QIAquick Gel Extraction kit (Qiagen, cat. no. 28704)
  • T4 DNA ligase
  • E. coli (TG1) competent cells (Lucigen, cat. no. 60502)
  • LB agar plates with 50 mg/liter kanamycin
  • Plasmid mini‐prep and maxi‐prep kits (Qiagen, cat. nos. 27106 and 12663)
  • HEK 293T cells
  • Dulbecco's modified Eagle medium (DMEM; Invitrogen, cat. no. 11995)
  • Fetal bovine serum (FBS)
  • 100× penicillin/streptomycin solution
  • Cell transfection kit (e.g., Lipofectamine, Invitrogen; XtrmeGene, Roche; Fugene HD, Promega) including:
    • Transfection reagent
  • Trypsin (0.25%, EDTA)
  • LB media with 50 mg/liter Kanamycin
  • Anhydrous sodium sulfate (Na 2SO 4)
  • 50‐ to 500‐ml round‐bottomed flasks and rubber septa
  • Water‐cooled condensation tube
  • Hot plate magnetic stirrer with contact thermometer oil bath
  • Ice bath
  • Fritted filter funnel
  • pH papers
  • Rotary evaporator
  • Columns for chromatography
  • Electronic oven
  • Dual nitrogen‐vacuum manifold with vacuum pump
  • Gel electrophoresis apparatus and power supply
  • 37°C E.coli incubator
  • 37°C incubator supplied with 5% CO 2
  • 10‐cm petri dishes for tissue culture
  • 8‐well chambered coverslip
  • Fluorescence microscope
  • Imaging acquiring and processing software (e.g., LAS AF software)
NOTE: Appropriate personal protective equipment (e.g., gloves, laboratory coats, goggles, and fume hoods) must be used for all synthetic work.

Alternate Protocol 1: Synthesis and Application of Covalent TMP‐Tag for Live‐Cell Imaging

  • L‐Aspartic acid 4‐tert‐butyl ester (CAS no. 3057‐74‐7)
  • Sodium carbonate
  • Acryloyl chloride
  • Sodium bisulfate (NaHSO 4)
  • Compound 2 TMP‐OH (from the protocol 1, step 2)
  • tert‐butyl N‐(3‐iodopropyl)carbamate (CAS no. 167479‐01‐8)
  • Cesium carbonate
  • Reverse‐phase C18 silica gel
  • 1‐Ethyl‐3‐(3‐dimethyllaminopropyl)carbodiimide hydrochloride (EDCI)
  • 4‐Dimethylaminopyridine (DMAP)
  • 6‐Carboxyfluorescein N‐hydroxysuccinimide ester (6‐carboxyfluorescein‐NHS ester; CAS no. 92557‐81‐8)
  • Triethylamine
  • Pyridine
  • Isobutyric anhydride
  • Toluene

Alternate Protocol 2: Synthesis and Application of Fluorogenic TMP‐Tag for Live‐Cell Imaging

  • Compound 9 TMP‐NH 2 (from protocol 2, step 8)
  • N‐Boc‐propargylglycine
  • Hydroxybenzotriazole (HOBt)
  • 1‐Ethyl‐3‐(3‐dimethyllaminopropyl)carbodiimide hydrochloride (EDCI)
  • Atto520‐NHS ester
  • N‐(3‐aminopropyl)‐N‐methylaniline
  • Di‐tert‐butyl dicarbonate
  • Fast Corinth V salt
  • Sodium carbonate (Na 2CO 3)
  • 3‐(Chlorosulfonyl)benzoyl chloride
  • 2‐Azidoethanol
  • Ascorbic acid
  • CuSO 4⋅5H 2O
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