Side-chain thioamides as fluorescence quenching probes

Abstract

Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing N^ε-thioacetyllysine and γ-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N^ε-thioacetyllysine into proteins expressed in E. coli using amber codon suppression.

Introduction

A thioamide is a single atom oxygen-to-sulfur substitution of an amide bond (hereafter also referred to as oxoamides for distinction from thioamides). Although a minimal substitution, the thioamide has distinct physico-chemical properties, including larger size, altered hydrogen bonding propensity and different photophysical properties.¹ Specifically, thioamides exhibit a significantly lower oxidation potential and a red-shifted π→π* absorption. The lowered oxidation potential (1.21 V for thioamides vs. 3.29 V for oxoamides; both vs. standard hydrogen electrode)² puts thioamides in the range of natural redox-active amino acids like tyrosine (1.4 V against standard hydrogen electrode).³ This allows thioamides to quench the fluorescence of a variety of dyes with appropriately matched reduction potentials through a photoinduced electron transfer (PeT) mechanism. The red-shifted π→π* absorption of 270 nm (vs. 200 nm for oxoamides)^4,5 allows for quenching of UV-fluorophores with spectral overlap between the thioamide absorption spectrum and the fluorophore emission spectrum through Förster resonance energy transfer (FRET).

Recently, thioamides have been used in a variety of biochemical contexts, including conformational stabilization of peptide macrocycles,⁶ conferring protease resistance to peptides,⁷ and as fluorescence turn-on probes for proteases.^8–10 However, it has also been shown that thioamide substitution in the backbone can have negative effects on protein stability.¹¹ Additionally, positioning of thioamides near the scissile bond of protease substrates must be evaluated carefully⁹. Moving the thioamide motif from the backbone to the side-chain of a peptide would provide an alternative approach to utilize thioamides, when backbone substitutions could be potentially disruptive. The side-chain thioamide N^ε-thioacetyllysine (Lys(Ac^S)) has been previously used as a non-hydrolyzable analogue of N^ε-acetyllysine (Lys(Ac)) for sirtuins, which represent Nicotinamide adenosine dinucleotide (NAD)-dependent class III histone deacetylase enzymes (HDACs).^12,13 γ-thioasparagine (Asn^γS) has previously been utilized in tripeptides as substrate to investigate the metal ion dependence of Oligosaccharyl Transferase.¹⁴

Here, we describe the assessment of fluorescence quenching properties of side-chain thioamides Lys(Ac^S) and Asn^γS in peptides. Through the synthesis and analysis of rigid polyproline ruler peptides of different lengths, we were able to quantify the distance dependent quenching of side-chain thioamides. Our results show that side-chain thioamides can provide a convenient alternative to backbone thioamides in protein folding or protease cleavage experiments.

Materials and Methods

General Information.

N^α-Fmoc-4-cyanophenylalanine was purchased from PepTech (Burlington, MA, USA). N^α-Fmoc-7-methoxycoumarin-4-yl-alanine (Fmoc-Mcm-OH) was purchased from Bachem (Basel, Switzerland). Piperidine was purchased from AmericanBio (Natick, MA, USA). Triisopropylsilane (TIPS) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). N^α-Fmoc-N^ε-acetyllysine, N^α-Boc-lysine, and N^ε-acetyllysine were purchased from ChemImpex (Wood Dale, IL, USA). Fmoc-Lys(Ac^S)-OH¹², Lys(Ac^S)-DBE¹⁵ and Fmoc-Asn^γS(Xan)-OH¹⁶ were synthesized as previously described. All other Fmoc protected amino acids and peptide synthesis reagents were purchased from EMD Millipore (Billerica, MA, USA). cOmplete Mini, EDTA-free protease inhibitor tablets were purchased from Roche Diagnostics (Mannheim, Germany). High-Density Nickel Agarose Beads were purchased from GoldBio (St. Louis, MO, USA). Amicon Ultra Spin Filters were purchased from EMD Millipore. Sephadex G-25 PD-10 desalting columns were purchased from GE Healthcare (Chicago, IL, USA). Acrylamide solution was purchased from Bio-Rad Laboratories (Hercules, CA, USA). [³⁵S]-Methionine was purchased from Dupont/NEN Research Products (Boston, MA, USA). All other reagents and solvents were purchased from Thermo-Fisher Scientific (Pittsburgh, PA, USA) or Sigma-Aldrich (St. Louis, MO, USA) and used without further purification unless otherwise specified.

High resolution electrospray ionization mass spectra (ESI-HRMS) were collected with a Waters LCT Premier XE liquid chromatograph/mass spectrometer (Milford, MA, USA). Low resolution electrospray ionization mass spectra (ESI-LRMS) were obtained on a Waters Acquity Ultra Performance LC connected to a single quadrupole detector (SQD) mass spectrometer. UV-Vis absorption measurements were performed on a Hewlett-Packard 8452A diode array spectrophotometer (currently Agilent Technologies; Santa Clara, CA, USA) or Thermo Fisher Scientific Genesys 150 UV/Vis Spectrophotometer (Waltham, MA, USA). Since OD₆₀₀ measurements are instrument specific (measured scattering intensity depends on the distance between sample and detector), values obtained on the Genesys 150 were multiplied by a factor of 1.5 (empirically determined) to match values obtained on HP 8452A. Fluorescence data were acquired on a Photon Technologies International (PTI) QuantaMaster40 fluorometer (currently Horiba Scientific, Edison, NJ, USA). Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker DRX 500 MHz instrument (Billerica, MA, USA). Matrix assisted laser desorption/ionization with time-of-flight detector (MALDI-TOF) mass spectra were acquired on a Bruker Ultraflex III or Microflex LRF instrument. Reverse-phase purification of small molecules was performed on a Biotage Isolera System on Biotage SNAP Ultra C18 columns (Charlotte, NC, USA). Analytical HPLC was performed on an Agilent 1100 Series HPLC system or on an Agilent 1260 Infinity II series UHPLC system (both Agilent, Santa Clara, CA, USA). Preparative HPLC was performed on a Varian Prostar HPLC system (currently Agilent Technologies. HPLC columns were purchased from W. R. Grace & Company (Columbia, MD, USA) or Phenomenex (Torrence, CA, USA).

General information about cloning.

All PCR reactions were carried out on a T100 thermocycler from Bio-Rad (Hercules, CA, USA). Primers and double stranded DNA fragments (gBlocks) were purchased from Integrated DNA Technologies (Coralville, IA, USA). dNTPs, SYBR Safe DNA gel stain and Agarose were purchased from Invitrogen (Waltham, MA, USA). Q5 High-Fidelity DNA polymerase, Q5 High-Fidelity DNA polymerase Master Mix, HiFi DNA Assembly Master Mix, Restriction Enzymes, competent High Efficiency E. coli cells (NEB 5-alpha and NEB 10-beta), T4 DNA Polynucleotide Kinase, T4 DNA Ligase and Monarch Gel Extraction Kit were purchased from New England Biolabs (Ipswich, MA, USA). Gel Green DNA gel stain was purchased from Biotium (Freemont, CA, USA). DNA Clean & Concentrator Kit and Plasmid Miniprep Kit were purchased from Zymo Research (Irvine, CA, USA). DNA concentrations were determined with a TECAN Nanoquant plate on an Infinite M1000Pro plate reader (Tecan, Männedorf, Switzerland). DNA sequencing (Sanger sequencing) was performed at the University of Pennsylvania DNA Sequencing Facility (Philadelphia, PA, USA). Agarose gels were visualized on a Typhoon Imager (GE Healthcare, Marlborough, MA, USA) or a SmartBlue transilluminator (Southern Labware, Cumming, GA 30028). pET His6-SUMO-TEV (2S-T) plasmid was a gift from Scott Gradia (Addgene plasmid # 29711; http://n2t.net/addgene:29711; RRID: Addgene_29711). pEVOL-mmPylRS plasmid was a gift from Prof. Wenshe Liu (Department of Chemistry, Texas A&M University, College Station, TX, USA). pET22b-T5 sfGFP and pET22b-T5 sfGFP(Y151Z) plasmids were a gift from Prof. Abhishek Chatterjee (Department of Chemistry, Boston College, Chestnut Hill, MA, USA).

Synthesis of Boc-Lys(Ac^S)-OH.

Boc-Lys-OH (492 mg, 2.0 mmoles) was suspended in 4.4 mL EtOH and 4.0 mL of a 10% (w/v) Na₂CO₃ solution were added. Ethyl dithioacetate (252 μL, 2.2 mmoles) was added and stirred overnight. On the next day, the solvent was removed in vacuo. The crude reaction mixture was dissolved in 10 mL H₂O and 3 M HCl was added dropwise while stirring until the solution was milky white and the pH was ~2. The product was extracted 3 times with CH₂Cl₂ before being dried over MgSO₄. After filtration, the solvent was removed in vacuo to yield the product as an orange foam (567 mg, 1.86 mmoles, 93.3%). ¹H NMR (500 MHz, Chloroform-d) δ 10.88 (s, 1H), 8.37 (s, 1H), 5.35 (d, J = 7.7 Hz, 1H), 4.07 (d, J = 75.4 Hz, 1H), 3.52 (s, 2H), 2.50 – 2.34 (m, 3H), 1.77 (s, 1H), 1.69 – 1.49 (m, 3H), 1.42 – 1.23 (m, 11H). ¹³C NMR (126 MHz, CDCl₃) δ 200.42, 176.09, 155.94, 81.99, 80.35, 77.42, 77.16, 76.91, 54.37, 52.99, 45.88, 33.50, 31.90, 28.16, 26.94, 22.68.

Synthesis of Lys(Ac^S)-OH.

Boc-Lys(Ac^S)-OH (567 mg, 1.86 mmoles) was dissolved in 3 mL CH₂Cl₂. 3 mL of trifluoroacetic acid (TFA) were added and the reaction was stirred at room temperature for 45 minutes, after which the solvent was removed in vacuo. The crude product was dissolved in 2 mL MeCN and precipitated with 90 mL of cold ether. The precipitate was pelleted by centrifugation at 4,000 RPM for 15 minutes and the supernatant was removed. The pellet was allowed to air dry for 15 minutes before being redissolved in 3 mL MeCN/H₂O (1:1) and dried by lyophilization. The product was obtained in high purity as white powder (542 mg, 1.70 mmoles, 91.6%). ¹H NMR (500 MHz, Deuterium Oxide) δ 3.77 (t, J = 6.1 Hz, 1H), 3.64 (t, J = 7.1 Hz, 2H), 2.52 (s, 3H), 2.02 – 1.84 (m, 2H), 1.80 – 1.67 (m, 2H), 1.55 – 1.37 (m, 2H). ESI⁺-HRMS calculated for C₈H₁₆N₂O₂SNa⁺: 259.2022; found: [M+Na]⁺: 259.2004.

Synthesis of (thio)acetyllysine containing peptides

Peptides were synthesized on 2-chlorotrityl resin (100–200 mesh, 10 μmol scale) in 3 mL fritted syringes. The resin was swelled in DMF for 20 minutes. All amino acids (5 equiv.) were dissolved in 2 mL DMF along with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (5 equiv.) and N,N-Diisopropylethylamine (DIPEA) (10 equiv.), except for the first amino acid of each peptide, for which HBTU was omitted, and Fmoc-Mcm-OH, for which only 2 equiv. amino acid/HBTU and 4 equiv. DIPEA were used. Amino acid couplings were carried out as double couplings for 30 minutes. Fmoc deprotections were carried out with 2% (v/v) 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF (3 × 2 min), After complete synthesis, the resin was washed with DCM and dried under vacuum before being cleaved off. The standard cleavage solution contained DCM/TFA/Triisopropylsilane (TIPS)/H₂O (10:8:1:1). After cleavage for 45 minutes, the solvent was removed in vacuo. The crude peptide was dissolved in 1 mL of 50% (v/v) MeCN in H₂O purified by HPLC. Fractions containing pure peptide were combined and lyophilized. HPLC retention times (Table S1), gradients (Table S2), and MALDI-TOF MS data (Table S3) for purified peptides are listed in the supporting information.

Synthesis of (thio)asparagine containing peptides

Peptides were synthesized on 2-chlorotrityl resin (100–200 mesh, 10 μmol scale) The resin was swelled in DMF for 20 minutes. All amino acids (5 equiv.) were dissolved in 2 mL DMF along with HBTU (5 equiv.) and DIPEA (10 equiv.), except for the first amino acid of each peptide, for which HBTU was omitted, and Fmoc-Asn^γS(Xan)-OH, for which only 2 equiv. amino acid and 4 equiv. DIPEA were used. Amino acid couplings were carried out as double couplings for 30 minutes. Fmoc deprotections were carried out with 20% (v/v) Piperidine in DMF (2 × 10 min), After complete synthesis, the resin was washed with DCM and dried under vacuum before being cleaved off. The cleavage solution contained 75% DCM, 10% TFA, 5% TIPS, 5% 1,2-Ethanedithiol (EDT) and 5% thioanisole (all percentages are v/v). After cleavage for 30 minutes, the solvent was removed in vacuo. The crude peptide was dissolved in 1 mL of 50% (v/v) MeCN in H₂O purified by HPLC. HPLC retention times (Table S1), gradients (Table S2), and MALDI-TOF MS data (Table S3) for purified peptides are listed in the supporting information.

Extinction coefficient measurements.

Extinction coefficients were determined for Cnf, Lys(Ac), and Lys(Ac^S) for accurate UV-Vis quantification. For each amino acid, a series of solutions were prepared in triplicate in phosphate buffered saline (10 mM Na₂HPO₄, 150 mM NaCl, pH 7.0, sterile filtered). Measurements of each sample were performed in duplicate and averaged. The measured absorbance values for each concentration were also averaged and plotted against the concentration. A linear regression with forced zero-intercept was performed. The slope of the regression corresponded to the extinction coefficient. The following values were determined. Cnf: ε₂₃₂ = 17,407 M⁻¹ cm⁻¹ ε₂₆₀ = 450 M⁻¹ cm⁻¹. Lys(Ac): ε₂₃₂ = 11 M⁻¹ cm⁻¹ ε₂₆₀ = 0 M⁻¹ cm⁻¹. Lys(Ac^S): ε₂₃₂ = 1,622 M⁻¹ cm⁻¹ ε₂₆₀ = 11,873 M⁻¹ cm⁻¹ ε₂₇₄ = 4,472 M⁻¹ cm⁻¹ ε₂₈₀ = 1,455 M⁻¹ cm⁻¹

Fluorescence measurements.

Peptide stocks were dissolved in 500 μL phosphate buffered saline (10 mM Na₂HPO₄, 150 mM NaCl, pH 7.0, sterile filtered) and quantified. The following extinction coefficients were used for determining peptide concentrations: Cnf/Lys(Ac) peptides: ε₂₃₂ = 17,418 M⁻¹ cm⁻¹; Cnf/Lys(Ac^S) peptides: ε₂₃₂ = 19,029 M⁻¹ cm⁻¹; Trp/Lys(Ac) peptides: ε₂₈₀ = 5,690 M⁻¹ cm⁻¹; Trp/Lys(Ac^S) peptides: ε₂₇₄ = 9,829 M⁻¹ cm⁻¹; Mcm peptides (same for Lys(Ac) and Lys(Ac^S)): ε₃₂₅ = 12,000 M⁻¹ cm⁻¹. Measurements were performed as triplicates at 10 μM concentration with the following settings: Cnf: excitation: 240 nm; emission: 260–400 nm; 5 nm slit widths; Trp: excitation: 278 nm; emission: 300–450 nm; 5 nm slit widths; Mcm: excitation: 325 nm; emission: 340–500 nm; 5 nm slit widths.

Small scale synthesis of Fmoc-Gln derivatives.

Synthesis for all derivatives was performed on a 50 μmol scale. A thionation solution was prepared by suspending P₄S₁₀ (134 mg, 300 μmoles) and Na₂CO₃ (31.8 mg, 300 μmoles) in 9 mL anhydrous tetrahydrofuran in an oven-dried 20 mL scintillation vial. The thionation mixture was stirred for 1 hour at room temperature under argon atmosphere. At the same time, reagents were weighed out in 1 dram vials. After the thionation mixture has stirred for 1 hour, 1.5 mL thionation solution (equals 1 equiv. of P₄S₁₀) was added to each vial. The solution was stirred for 1 minute to dissolve amino acids and blanketed with Argon. After 30 minutes a sample was taken for HPLC analysis (10 μL sample added to 1 mL MeOH). Reactions were blanketed again with Argon before being closed, sealed with parafilm and stirred for 20 hours at room temperature. After 20 hours, another sample was taken and was analyzed by HPLC and LCMS. HPLC analysis was done using 20 μL injections on a Luna Omega C18 analytical column using gradient O (see Table S2).

Synthesis of Fmoc-Gln^δS(Trt)-OH.

P₄S₁₀ (233 mg, 0.5 mmoles) and Na₂CO₃ (53.0 mg, 0.5 mmoles) were suspended in 10 mL anhydrous tetrahydrofuran. The solution was stirred under argon atmosphere for 1 hour. Fmoc-Gln(Trt)-OH (122 mg, 0.2 mmoles) were added and the reaction was stirred under argon atmosphere overnight. The next day the solvent was removed in vacuo. The crude product was redissolved in 1 mL MeOH and purified on a Biotage system using a SNAP Ultra C18 column (12 g) on a gradient from 50%−100% Solvent B (0.1% TFA in acetonitrile) over 20 column volumes. Fractions containing product were lyophilized and the product obtained as a white powder in low yield (22.0 mg, 35.1 μmoles, 17.6%). ¹H NMR (500 MHz, Chloroform-d) δ 7.77 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.34 – 7.29 (m, 8H), 7.29 – 7.27 (m, 2H), 7.26 (d, J = 2.8 Hz, 1H), 7.26 – 7.21 (m, 6H), 6.84 (s, 1H), 6.02 (d, J = 7.6 Hz, 1H), 4.57 (dd, J = 10.6, 6.8 Hz, 1H), 4.36 (dd, J = 10.7, 7.2 Hz, 1H), 4.30 – 4.21 (m, 2H), 2.52 – 2.39 (m, 2H), 2.22 (d, J = 15.3 Hz, 1H), 2.04 – 1.83 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 200.28, 171.33, 156.40, 144.50, 143.90, 141.47, 128.78, 128.18, 127.89, 127.87, 127.31, 127.27, 125.27, 120.12, 71.03, 67.31, 61.84, 47.36, 33.18, 27.30. ESI⁺-HRMS calculated for C₃₉H₃₅N₂O₄S⁺: 627.2318; found: [M+H]⁺: 627.2313.

In vitro translation and suppression.

We translated an mRNA that encodes a 173-amino acid protein (derived from Kv1.3, see Po et al.¹⁷), which was engineered to contain an amber codon at position 118 of the nascent chain and an enzyme restriction site at 173. The latter cut-site was used to generate mRNA lacking a stop codon so the nascent chain remains attached to tRNA and the ribosome. Translation reaction mixtures (25μL total) contained rabbit reticulocyte lysate, [³⁵S]-methionine (2μl/25μl translation mixture; ~10 μCi/μl Express), and RNasin according to Promega Protocol and Application Guide. All samples were pre-incubated for 5 min with [³⁵S]-methionine and mRNA (2 μL of a 0.4μg/μl mRNA). Addition of either unaminoacylated tRNA^amber (control: tRNA^amber present in Flexizyme reaction in the absence of a DBE-activated Lys(Ac^S)) or suppressor Lys(Ac^S)-tRNA^amber was added to the translation reaction and translation was continued for 90 min. Total amber tRNA concentration was ~ 20 uM.

Translation reactions (25 μL) were added to 500 μL phosphate-buffered saline, PBS*, comprised of PBS, Ca-free, containing 4 mM MgCl₂, 2 mM DTT, pH 7.3, and centrifuged at 70,000 rpm/20 min/4°C (TLA 100.3 Beckman rotor) through a sucrose cushion (120 μL, containing 0.5 M sucrose, 100 mM KCl, 50 mM HEPES, 5 mM MgCl₂, 2 mM DTT, pH 7.5). Pellets were resuspended in 30μL of LDS NuPAGE Sample Buffer, RNase (final concentration of 20 ng/μL), and incubated at room temperature for 30 minutes. Samples were fractionated on NuPAGE 10% Bis-Tris gels. Quantitation of gels was carried out directly using a Molecular Dynamics (Sunnyvale, CA) Typhoon FLA 9500 PhosphorImager and ImageQuant. The fraction of full-length protein (FL) was calculated as the ratio of FL band to the sum of the FL and arrested at amber (“Arr”). N.B.: The amber-arrested peptide band (~12 kD) has 3 methionines and the FL peptide (~ 17kD) has 4 methionines. Correction for the methionine difference was included in the calculation of fraction of FL.

General procedure for PCR.

Primers were dissolved in Milli-Q grade water to be at a concentration of 50 μM. The amount of plasmid used ranged from 0.1 – 1 ng total DNA. Primers, plasmid and buffer components were mixed according to manufacturer recommendations. PCR was carried out for 35 cycles at annealing temperatures calculated with the New England Biolabs Tm calculator tool (https://tmcalculator.neb.com). Alternatively, a ‘Touchdown protocol’ was used, where annealing started with 70 °C in the first cycle and was reduced in each of the 19 subsequent cycles by 0.5 °C each, followed by 15 rounds of amplification at 55 °C.

Following amplification PCR product was isolated using a DNA Concentrator & Cleanup Kit. PCR product was quantified to verify successful PCR amplification. PCR product was circularized by mixing 2 μL PCR product with 5 μL Milli-Q grade water, 1 μL 10x T4 DNA Ligase Buffer, 1 μL T4 Polynucleotide Kinase, 0.5 μL T4 DNA Ligase and 0.5 μL DpnI. This mixture was incubated at 37 °C for 45 minutes, cooled on ice and 50 μL of competent NEB 5-alpha cells were added. Cells were transformed and plated on an LB-Agar plate containing appropriate antibiotic. Individual colonies were picked, grown up in 5 mL LB-medium and their DNA was extracted using a Plasmid Miniprep Kit according to manufacturer’s protocol. Quantified DNA was submitted for sanger sequence analysis to verify correct sequence. Sequencing primer was provided by Sequencing Center and aligns with T7 promoter unless otherwise noted.

General procedure for Gibson Assembly.

Primers were dissolved in Milli-Q grade water to be at a concentration of 50 μM. The amount of plasmid used ranged from 0.1 – 1 ng total DNA. Vector and insert were amplified simultaneously using a ‘Touchdown protocol’, where annealing started with 70 °C in the first cycle and was reduced in each of the 19 subsequent cycles by 0.5 °C each, followed by 15 amplifications at 55 °C. PCR product was purified using a 1% Agarose gel containing SYBR Safe or GelGreen Dye. The gel was visualized using a Blue light transilluminator and bands of the expected size were excised. DNA was extracted using a DNA gel extraction kit according to manufacturer’s protocol and isolated DNA was quantified. DNA assembly was performed with 100 ng of vector DNA and 2 equiv. of insert DNA (5 equiv. for insert <300 bp) using a HiFi DNA Assembly Mix according to manufacturer’s protocol. 2 μL of the ligated product were transformed in 50 μL competent NEB10β cells according to manufacturer’s instructions and plated on an LB-Agar plate containing appropriate antibiotic. Individual colonies were picked, grown up in 5 mL LB-medium and their DNA was extracted using a Plasmid Miniprep Kit according to manufacturer’s protocol. Quantified DNA was submitted for sanger sequence analysis to verify correct sequence. Sequencing primer was provided by Sequencing Center and aligns with T7 promoter unless otherwise noted.

Cloning of pEVOL-chAcK3RS.

The plasmid was constructed using Gibson Assembly. The vector donor plasmid was pEVOL-PylRS and was a present from Prof. Wenshe Liu. The insert sequence was obtained from the published amino acid sequence for chAcKRS3¹⁸ and is shown in List S1. The sequence was optimized for E. coli using IDT’s codon optimization tool and was purchased as gBlock from IDT (for DNA Sequence see List S1). Sequencing was submitted with custom sequencing primers from 3’ and 5’ end to cover the whole range of interest.

• Forward primer for vector:

• 5’ – ACCAACCTGT AAGTCGAC – 3’

• Reverse complement primer for vector:

• 5’ – CATACTAGTA ATTCCTCCTG TTAGC – 3’

• Forward primer for sequencing:

• 5’ – CTACCTGACG CTTTTTATCG CA – 3’

• Reverse complement primer for sequencing:

• 5’ – TTTATCAGAC CGCTTCTGCG TT – 3’

Cloning of pET sfGFP.

The plasmid was constructed using Gibson Assembly. The vector donor plasmid was pET His6-SUMO-TEV. The insert donor plasmid was pET22b-T5-sfGFP and was a gift from Prof. Abhishek Chatterjee (for sequence see List S1). Primers were designed to introduce 10 nt overlaps, i.e. the 5’ and 3’ and of insert contained 10 nt of the vector and vice versa. This created the necessary 20 nt overlap for Gibson assembly.

• Forward primer for vector:

• 5’ – CCACCACTAA CGGATCCGCG ATCG – 3’

• Reverse complement primer for vector:

• 5’ – CTTTGCTCAT ATGTATATCT CCTTCTTAAA GTTAAACAAA ATTATTTCTA G– 3’

• Forward primer for insert:

• 5’ – AGATATACAT ATGAGCAAAG GAGAAGAAC – 3’

• Reverse complement primer for insert:

• 5’ – CGCGGATCCG TTAGTGGTGG TGGTGGT – 3’

Cloning of pET sfGFP(Y₁₅₁Z).

The plasmid was constructed using Gibson Assembly. The vector donor plasmid was pET His6-SUMO-TEV -EcoR1. The insert donor plasmid was pET22b-T5-sfGFP(Y₁₅₁Z) and was a gift from Prof. Abhishek Chatterjee (for sequence see List S1). Primers were designed to introduce 10 nt overlaps, i.e. the 5’ and 3’ and of insert contained 10 nt of the vector and vice versa. This created the necessary 20 nt overlap for Gibson assembly.

• Forward primer for vector:

• 5’ – CCACCACTAA CGGATCCGCG ATCG – 3’

• Reverse complement primer for vector:

• 5’ – CTTTGCTCAT ATGTATATCT CCTTCTTAAA GTTAAACAAA ATTATTTCTA G– 3’

• Forward primer for insert:

• 5’ – AGATATACAT ATGAGCAAAG GAGAAGAAC – 3’

• Reverse complement primer for insert:

• 5’ – CGCGGATCCG TTAGTGGTGG TGGTGGT – 3’

Expression of sfGFP (WT or Y151Z).

A pET plasmid containing sfGFP (WT) was transformed into BL21 (DE3) cells and plated on LB-Agar plates containing Ampicillin (100 μg/mL). pET plasmid containing sfGFP (Y₁₅₁Z) and pEVOL-chAcK3RS were co-transformed into BL21 (DE3) cells and plated on LB-Agar plates containing Ampicillin (100 μg/mL) and Chloramphenicol (25 μg/mL). Non-inducing medium (NIM) and autoinducing medium (AIM) containing Lactose and Arabinose were prepared as described by Mehl and coworkers¹⁹, except for the use 25x M-salts instead of 50x M-salts (to extend shelf life). A single colony was picked and grown in 3 mL LB media containing Ampicillin (100 μg/mL) for WT sfGFP expression or Ampicillin (100 μg/mL) and Chloramphenicol (25 μg/mL) for sfGFP (Y₁₅₁Z) expression at 37 °C with 250 RPM shaking overnight. Next day, 25 mL cultures with AIM in 125 mL Erlenmeyer flasks were prepared by adding 20 mM nicotinamide, 10 mM Lys(Ac) or Lys(Ac^S), and antibiotics (Ampicillin (100 μg/mL) for WT sfGFP expression or Ampicillin (100 μg/mL) and Chloramphenicol (25 μg/mL) for sfGFP (Y₁₅₁Z) expression). Secondary cultures were inoculated with 0.5% (v/v) overnight culture and grown at 37 °C with 250 RPM shaking for 24 hours. At this point for each culture three 1:10 dilutions (1 mL each) were prepared and OD₆₀₀ and fluorescence (exitation: 395 nm; emission: 510 nm; 2 nm slit width, 0.1 sec integration time) measured in triplicates. For SDS-PAGE analysis, cells from 500 μL were pelleted by spinning at 13.2 kRPM in a tabletop microcentrifuge for 3 minutes. The supernatant was discarded and cell pellet frozen until further use. When needed, cell pellets were thawed on ice and resuspended in 50 μL 1x LDS buffer containing 200 mM DTT and sample was boiled at >90 °C for 5 minutes before loading 15 μL sample.

Cells were harvested by centrifugation at 4,000 RPM in a GS3 rotor and Sorvall RC-5 centrifuge for 20 min at 4 °C. The supernatant was discarded and the cell pellet was suspended in 5 mL lysis buffer (40 mM Tris, 500 mM NaCl, 1 mM PMSF, pH 8.3) containing a broad-spectrum protease inhibitor. Resuspended cells were then lysed on ice by sonication (30 amps power, 1 second pulse, 2 second rest, 3 minutes total sonication time) and then pelleted at 14,000 RPM in an SS-34 rotor (Sorvall RC-5 centrifuge) for 30 min at 4 °C. In the meantime, 1 mL (settled volume) of Nickel Agarose beads were washed with 5 mL equilibration buffer (50 mM HEPES, pH 7.5). The crude cell lysate was incubated with the washed nickel resin for 1 h on ice with shaking. The slurry was then added to a fritted syringe and the liquid was allowed to flow through. The resin was then washed with 5 mL of equilibration buffer, 5 mL of wash buffer 1 (50 mM HEPES, 5 mM imidazole, pH 7.5), 5 mL wash buffer 2 (50 mM HEPES, 50 mM imidazole, pH 7.5) before being eluted from the resin in 2.5 mL of elution buffer (50 mM HEPES, 300 mM imidazole, pH 7.5). The crude protein was buffer exchanged using PD-10 desalting columns equilibrated with 10 mM NH₄HCO₃ according to manufacturer’s instruction (gravity protocol) and stored at −20 °C until further use.

Results and Discussion

While substitution of amide linkages in backbones has been pursued for decades,²⁰ the incorporation of thioamides into side-chains of amino acids is a newer endeavor. Incorporation of side-chain thioamides was previously achieved using solution phase peptide synthesis.¹⁴ However, for the synthesis of longer peptides, solid-phase peptide synthesis (SPPS) is necessary. Synthesis of side-chain bearing thioamide building blocks for SPPS is synthetically more challenging and wasn’t reported until 2000.¹⁶ We envisioned utilizing three different amino acids with thioamide bearing side-chains. The most obvious choice is the use of thionated analogues of the natural amide-containing amino acids asparagine or glutamine. This would result in the amino acids γ-thio-asparagine (Asn^γS) and δ-thio-glutamine (Gln^δS). We also envisioned using N^ε-thioacetyllysine (Lys(Ac^S)), as a thionated analogue of a post-translationally modified amino acid¹².

We began our efforts with the synthesis of a series of peptides containing Lys(Ac^S). We synthesized polyproline “ruler” peptides as rigid spacers with a fluorophore on one end and a thioamide on the other. Additionally, we also synthesized the corresponding oxoamide versions of all peptides as references. Our laboratory has previously utilized the same approach to assess distance dependence fluorescence quenching by backbone thioamides²¹. We chose three different fluorophores based on their interaction with thioamides: The fluorescent emission spectra of p-Cyanophenylalanine (Cnf) has sufficient spectral overlap with the absorption spectrum of thioamides to be quenched through a FRET based mechanism. 7-Methoxycoumarinyl-2-alanine (Mcm), on the other hand, does not have spectral overlap and relies on quenching through a PeT based mechanism. Tryptophan acts as a mixed case: it has a low enough reduction potential to be quenched through PeT, but also exhibits sufficient spectral overlap to interact with thioamides through a minimal FRET based mechanism.

Our general design consisted of Lys(Ac^S) or Lys(Ac) placed at the C-terminus and a fluorophore at the N-terminus. These two residues were separated by 2–6 proline residues, which act as a rigid ruler. The synthetic incorporation of Lys(Ac^S) into peptides using solid phase peptide synthesis (SPPS) has been described previously in the literature¹². One should be aware that the use of piperidine solutions for Fmoc deprotections should be avoided when using Fmoc-Lys(Ac^S), as it can lead to formation of an amidine byproduct.²² The peptides were dissolved at 10 μM concentration in phosphate buffered saline (10 mM Na₂HPO₄, 150 mM NaCl, pH 7.0) and their fluorescence emission was measured. Fluorescence intensity from thioamide containing peptides was normalized to the corresponding oxoamide peptide and plotted in Figure 1. All thioamide/fluorophore pairs show a strong distance-dependence in their quenching efficiency, with Trp showing the strongest effect, especially in the range of 2–4 proline residues. Cnf is quenched with an almost linear dependence of quenching efficiency on distance. Although Mcm can only be quenched through PeT based mechanisms, which occur on short distances, they still seem to quench fluorescence in the longest peptides investigated, which cannot be explained based on simple models of the polyproline segment as a rigid spacer. Ongoing analysis of the data along with computational modeling of this peptide series is currently underway.

Figure 1:

Fluorescence quenching by side-chain thioamide N^ε-thioacetyllysine. Top: Structure of polyproline rulers with various fluorophores. Bottom: Quenching efficiency of peptides with various fluorophores (normalized to corresponding oxo peptide). The number of proline residues between fluorophore and quencher are indicated by n.

The synthesis and incorporation of the other two side-chain thioamides proved more challenging. Fmoc-protected precursors of Asn^γS for SPPS have been synthesized previously, but not successfully incorporated into peptides.¹⁶ Synthesis of Fmoc-protected Gln^δS has been attempted but was not successful.¹⁶ We were able to recapitulate the findings of Sanderson et al., where we were able to synthesize Fmoc-Asn^γS(Xan)-OH based on their synthetic route, but not the analogous Fmoc-Gln^δS(Xan)-OH. However, while Sanderson et al. were successful in the synthesis of the protected monomer, they were unsuccessful in their attempts to isolate the desired peptides from resin using standard cleavage conditions with 95% TFA. We have previously observed that thioamide-containing backbone can decompose under these conditions.²³ Reducing TFA concentrations to less than 50% or lower and reducing cleavage times to one hour or less can significantly increase the yield of thioamide-containing peptides post purification.

To qualitatively demonstrate the utility of Asn^γS as a fluorescence quencher probe, we synthesized a small series of peptides similar to the ones above. In these peptides, Cnf was the N-terminal residue and Asn or Asn^γS was the C-terminal residue. The fluorophore/quencher pair was separated by either 2, 4, or 6 proline residues. With our modified, mild peptide cleavage conditions, we were able to cleave the peptides off the resin and isolate pure Asn^γS containing peptides. Fluorescence measurements of concentration-matched peptides made as described above showed that Asn^γS was indeed capable of quenching fluorescence in a distance dependent manner that was similar to that of Lys(Ac^S) (see Figure 2).

Figure 2:

Distance dependent fluorescence quenching of Asn^γS is in good agreement with data obtained from Lys(Ac^S) measurements. Fluorescence intensity is normalized to corresponding oxo peptide. Peptides with n=3 and 5 were not synthesized for Asn^γS. The number of proline residues between fluorophore and quencher are indicated by n.

We then investigated whether an Fmoc-protected Gln^δS monomer might be obtained through direct thionation with phosphorous pentasulfide (P₄S₁₀) of commercially available derivatives. This would drastically reduce the number of steps needed to synthesize precursors. We hypothesized that the size of the side-chain protecting group might influence thionation efficiency as large protecting groups might sterically obstruct the thionation site. We attempted direct thionation of five differently protected Gln derivatives. Small scale (50 μmol each) reactions were set up and analyzed after 20 hours by high performance liquid chromatography (HPLC) and liquid chromatography coupled mass spectrometry (LC-MS). As shown in Figure 3, direct thionation was successful with all tested derivatives. While 2,4,6-trimethoxybenzyl- (Tmob) and 4,4’-dimethyoxybenzhydryl-protected (Mbh) Fmoc-Gln showed the highest rate of conversion (defined as consumption of starting material), they also showed additional peaks that had masses corresponding to species with multiple thionations. Based on these results, we proceeded to set up a larger scale synthesis with a trityl (Trt) side-chain protecting group. We chose Trt because it had similar clean conversion as xanthyl (Xan) protected Gln, but exhibited significantly better solubility. While the isolated yield was rather low, it is comparable to our overall yields obtained from the multistep synthesis of Fmoc-Asn^γS(Xan)-OH and is to our knowledge the first successful synthesis of an Fmoc protected precursor for Gln^δS. Unfortunately, attempts to incorporate Gln^δS into peptides using conditions similar to Asn^γS failed. Nevertheless, the synthetic accessibility of Gln^δS derivatives through direct thionation is a noteworthy achievement. Since synthesis of other Fmoc-protected thioamide building blocks for SPPS usually requires multiple steps, this single step reaction is a big improvement. While we were not able to incorporate Fmoc-Gln^δS(Trt)-OH into peptides, it is possible that changing the amino acid position in the peptide or switching the side-chain protecting group might result in successful incorporation. This work however goes beyond the scope of this manuscript.

Figure 3:

Direct thionation of Fmoc-Gln with various side-chain protecting groups. Analytical HPLC traces shown. # indicates peak with mass corresponding to monothionation. Abbreviations: Dmcp = Dimethylcyclopropyl; Tmob = 2,4,6-Trimethoxybenzyl; Mbh = 4,4’-dimethyoxybenzhydryl; Xan = Xanthyl; Trt = Trityl.

Recently, Söll, Miller, and coworkers successfully incorporated Lys(Ac^S) into proteins using Flexizyme combined with in vitro translation.¹⁵ Inspired by their work, we attempted in vivo incorporation of Lys(Ac^S). Using the methods described by Söll et al., we synthesized N^ε-thioacetyllysine dinitrobenzylester (Lys(Ac^S)-DBE). We used the catalytic flexizyme RNA to aminoacylate tRNA^amber with Lys(Ac^S)-DBE as substrate. Using the resulting Lys(Ac^S)-tRNA^amber in an in vitro translation experiment, we were able to incorporate Lys(Ac^S) (see SI, Fig S1). We used [³⁵S]-Methionine radiolabels for detection. While the amounts of proteins we obtained were too low for analysis by mass spectrometry to verify the identity of the resulting peptide, Söll, Miller, and coworkers convincingly demonstrated Lys(Ac^S) incorporation. Thus, we conclude that Lys(Ac^S) incorporation using in vivo translation seems to be a robust approach to obtain small quantities of Lys(Ac^S) containing proteins. These amounts would be sufficient for single-molecule FRET applications of thioamides using brighter fluorophores such as Alexafluor 488 which we have shown to be quenched by thioamides.²⁴

To obtain larger amounts of protein, we attempted in vivo incorporation using an orthogonal tRNA synthetase/tRNA pair and amber codon suppression in E. coli. Lys(Ac) is a surprisingly challenging unnatural amino acid to incorporate and often suffers from low yields. We decided to use a pyrrolysyl-based synthetase developed in collaboration between the Söll and Liu labs using phage-assisted continuous evolution (PACE).¹⁸ This particular synthetase (chAcK3RS) is currently one of the best performing synthetases for the incorporation of Lys(Ac).

We chose super-folder green fluorescent protein (sfGFP) as our test system. It is a well-established test system for unnatural amino acid (Uaa) incorporation. Premature truncation at the amber stop codon site results in a shortened sfGFP (sfGFP1–150) fragment that has the amino acids needed for chromophore maturation, but is unable to fold properly and is therefore non-fluorescent. Proteins were expressed for 24 hours in autoinducing medium containing 20 mM nicotinamide to suppress endogenously expressed CobB, a NAD-dependent lysine deacylase²⁵. After 24 hours, optical density at 600 nm (OD₆₀₀) and fluorescence of the crude suspension was measured before harvesting and purifying cells. As shown in Figure 4, cells produced significantly lower amount of protein (estimated by fluorescence), especially if one accounts for the number of cells (estimated by OD₆₀₀): Only 9.0% of WT sfGFP fluorescence was observed under Lys(Ac) incorporation conditions. The measured fluorescence for Lys(Ac^S) incorporation was roughly 1/3 of the corresponding oxo version (4.0%) and only slightly above the negative control without Uaa (3.6%), which accounts for non-specific readthrough of the amber codon.

Figure 4:

Fluorescence (left) and OD600 (right) measurements (in triplicate) of attempts to incorporate Uaas into sfGFP in cells grown for 24 hours in autoinducing medium. Media for non-WT expressions contained 10 mM Lys(Ac) or Lys(Ac^S).

To identify whether the decrease in observed fluorescence was due to lower amounts of full-length protein or if incorporated Lys(Ac^S) could partially quench sfGFP fluorescence, we harvested the cells and isolated the protein using nickel affinity chromatography. Purified proteins were desalted and further analyzed by trypsin digests. Results from gel electrophoresis (SDS-PAGE) and tryptic digest are shown in Figure S2. SDS-PAGE of isolated proteins indicated that the decrease in fluorescence was in fact due to lower amounts of protein. Analysis of the digested proteins revealed that Lys(Ac) was incorporated, even when Lys(Ac^S) was provided in the cell culture medium. The mechanism of this S-to-O exchange is unclear. The in vitro translation experiment from Söll et. al¹⁵ showed that the ribosome is able to incorporate Lys(Ac^S) once the tRNA is aminoacylated and that it is possible to isolate thioamide-containing proteins. Our hypothesis is that Lys(Ac^S) undergoes an S-to-O exchange in vivo prior to incorporation. The resulting Lys(Ac) can get incorporated into proteins, but its concentration is significantly lower than in the corresponding control experiment, hence leading to a lower protein yield. However, further systematic investigation would be necessary to elucidate the exact mechanism.

Conclusion:

Here we synthesized Fmoc-protected precursors of N^ε-thioacetyllysine, γ-thioasparagine, and δ-thioglutamine. We were able to incorporate N^ε-thioacetyllysine and γ-thioasparagine into rigid model peptides and showed that these side-chain thioamides exhibit distance dependent quenching through FRET and PeT. Although we were not able to incorporate δ-thioglutamine into peptides, we were able to develop a concise synthesis for its Fmoc-protected monomer. This will allow us to synthesize the amino acid with other protecting groups and re-attempt incorporation in the future with other protecting groups. While ribosomal incorporation of N^ε-thioacetyllysine into proteins was successful using an in vitro translation system, in vivo incorporation using amber codon suppression failed. In a recently published study, Pless and coworkers were successful in incorporating Lys(Ac^S) containing peptides into full length proteins in eukaryotic cells using in vivo tandem protein trans-splicing.²⁶ While our synthetase-mediated incorporation experiments lead us to believe that Lys(Ac^S) undergoes S-to-O exchange in cells, it is likely that this process occurs at the amino acid level and that our synthetase is selective for Lys(Ac) over Lys(Ac^S), which drives Lys(Ac) incorporation into proteins. A synthetase designed or evolved to be selective for Lys(Ac^S) could enable synthetase-mediated in vivo incorporation. Additionally, the existing synthetase could be used to scale up the yields of in vitro translation reactions which have been shown to be robust to S-to-O exchange since these yields are typically limited by the levels of semi-synthetic aminoacyl tRNA.²⁷ Altogether, our research shows that side-chain thioamides are viable options for fluorescence quenching experiments in cases where backbone thioamides are disruptive to the protein structure and that they have the potential for incorporation into full-length proteins.