Click chemistry reagent; unnatural amino acid

Modified soluble superfolded green fluorescent protein (sfGFP) containing 4-azido-L-phenylalanine or p-propargyloxy-L-phenylalanine (pPaF) would accumulate 0.9-1.7 mg/mL in CFPS solutions when using a cell-free protein synthesis (CFPS) technique [1].

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In this study, pAzF was used for site-specific incorporation into superfolder green fluorescent protein (sfGFP) using a cell-free protein synthesis platform. Using the new method (simultaneous production of orthogonal tRNA and modified protein), 0.9–1.7 mg/mL of modified soluble sfGFP containing either pAzF or pPaF accumulated after 16 h, corresponding to 50–88% suppression efficiency. The specific activities and soluble yields of pAzF-containing sfGFP proteins were similar to those containing pPaF at the tested sites [1].

The fluorescence of sfGFP containing pAzF at position 66 was abolished; substitution at position 92 reduced fluorescence by 60–70%; other substitutions did not alter specific activity substantially (reduced by ≤20%) [1].

Twenty-five-microlitre PANOx SP reaction solutions contained, unless otherwise noted: 10 mM ammonium glutamate, 20 mM magnesium glutamate, 175 mM potassium glutamate, 1.2 mM ATP, 0.86 mM each of CMP, GMP and UMP, 10 mM dibasic potassium phosphate, 34 μg/ml folinic acid, 171 μg/ml E. coli tRNAs, 33 mM phosphoenolpyruvate (PEP), 1.5 mM spermidine, 1 mM putrescine, 0.33 mM nicotinamide adenine dinucleotide (NAD), 0.27 mM coenzyme A, 2.7 mM sodium oxalate, 2 mM each of the 20 amino acids, 5 μM l-[14C(U)]-leucine, 2 mM p-azido-l-phenylalanine (pAzF) or 4 mM p-propargyloxy-l-phenylalanine (pPaF), 0.3 mg/ml pAzFRS or 0.5 mg/ml pPaFRS (prepared as described above), 100 μg/ml T7 RNA polymerase, 6 nM sfGFP plasmid, 0.2 mg/ml linearized o-tRNA or o-tRNAopt plasmid template and either 0.24 volume of standard KC6 extract or 0.28 volume of orthogonal extract. The glutamate salts were diluted from a 10-fold concentrated (10×) solution. Similarly, a 10× NTMP Master Mix solution contained ATP, the three NMPs, folinic acid and the E. coli tRNAs, and was adjusted to pH 7.3 with dibasic potassium phosphate. The natural amino acids were diluted from a stock solution, which contained a 50 mM concentration of each amino acid. This stock solution was prepared by adding the amino acids in the following order (given in their one-letter code): R, V, W, F, I, L, C, M, A, N, D, E, G, Q, H, K, P, S, T, Y. During the preparation, it was ensured that each amino acid was dissolved before addition of the next, except tyrosine, which is added last and remains suspended in the solution. The reactions were incubated at 30°C for 16 h, unless stated otherwise. CFPS reactions containing pAzF were prepared in a dark room, and the tubes containing the reaction solutions were wrapped in aluminum foil to prevent photodissociation of the aromatic azide[1].

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The orthogonal S30 cell extract was prepared from KC6 cells harbouring the pDule-tRNA plasmid, as described. The lysate was not diluted after homogenization. The specific growth rate of this KC6/o-tRNA culture was 0.57 h−1 during the batch fermentation. The standard KC6 extract was prepared as described. After homogenization, 0.2 ml S30 buffer (14 mM magnesium acetate, 60 mM potassium acetate, 10 mM Tris acetate, pH 8.2) was added per ml of lysate, and the diluted lysate was dialysed against 100 volumes of S30 buffer at 4°C four times for 30 min each. Finally, the S30 cell extract was centrifuged at 10 000g and 4°C for 20 min, and the supernatant was aliquoted, flash-frozen and stored at −80°C. The specific growth rate of the KC6 culture was 0.96 h−1 during the fed-batch fermentation[1].

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Cell-free protein synthesis (CFPS) reactions were performed using the PANOx SP system. Reaction solutions (25 μL) contained: 10 mM ammonium glutamate, 20 mM magnesium glutamate, 175 mM potassium glutamate, 1.2 mM ATP, 0.86 mM each of CMP, GMP and UMP, 10 mM dibasic potassium phosphate, 34 μg/mL folinic acid, 171 μg/mL E. coli tRNAs, 33 mM phosphoenolpyruvate (PEP), 1.5 mM spermidine, 1 mM putrescine, 0.33 mM nicotinamide adenine dinucleotide (NAD), 0.27 mM coenzyme A, 2.7 mM sodium oxalate, 2 mM each of the 20 natural amino acids, 5 μM [14C(U)]-leucine, 2 mM p-azido-L-phenylalanine (pAzF), 0.3 mg/mL pAzFRS (synthetase specific to pAzF), 100 μg/mL T7 RNA polymerase, 6 nM sfGFP plasmid, 0.2 mg/mL linearized orthogonal tRNA (or o-tRNAopt) plasmid template, and either 0.24 volume of standard KC6 extract or 0.28 volume of orthogonal extract. Reactions were incubated at 30°C for 16 h. CFPS reactions containing pAzF were prepared in a dark room and tubes were wrapped in aluminum foil to prevent photodissociation of the aromatic azide [1].

Modified sfGFP proteins containing pAzF were quantified by radiolabeling with [14C]-leucine. Total and soluble protein concentrations were measured by trichloroacetic acid precipitation and scintillation counting. Full-length protein amounts were determined by densitometry analysis of SDS-PAGE autoradiograms. Suppression efficiency was defined as the ratio of full-length modified protein yield to that of natural sfGFP [1].

For fluorescence characterization, sfGFP proteins (natural or modified with pAzF at various positions) were produced in CFPS, purified via Strep-Tactin affinity chromatography, and dialyzed. Fluorescence was measured between 523–548 nm with excitation at 485 nm, and specific activity (AU/ng) was calculated by dividing fluorescence by protein concentration [1].

[1]. Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation. Nucleic Acids Res. 2013 Jun;41(11):5949-63.

4-Azide-L-phenylalanine is a 4-azidophenylalanine with an L-configuration. It can be used as a bioorthogonal click chemistry reagent. It is a derivative of 4-azidophenylalanine and L-phenylalanine, and also an enantiomer of 4-azido-D-phenylalanine.

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p-Azido-L-phenylalanine (pAzF) is a non-natural amino acid containing an azide moiety. It can be site-specifically incorporated into proteins using an orthogonal tRNA/aminoacyl-tRNA synthetase pair derived from Methanocaldococcus jannaschii. Proteins containing pAzF can be directly coupled using copper(I)-catalyzed azide-alkyne cycloaddition (Click chemistry) [1].

In this study, a new cell-free platform was developed where the orthogonal tRNA is synthesized simultaneously with the modified protein using a hammerhead ribozyme (transzyme approach). This method achieved high yields (0.9–1.7 mg/mL) of sfGFP containing pAzF or pPaF, with suppression efficiencies of 50–88%. The o-tRNA could be produced from a linearized plasmid or a crude PCR product. The platform is modular and can be used to incorporate >30 non-natural amino acids [1].

C9H10N4O2

206.205

206.08

C, 52.42; H, 4.89; N, 27.17; O, 15.52

33173-53-4

4-Azido-L-phenylalanine hydrochloride;34670-43-4

3080772

Typically exists as white to yellow solids at room temperature

1.735

2

5

4

15

267

1

O([H])C([C@]([H])(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N=[N+]=[N-])N([H])[H])=O

NEMHIKRLROONTL-QMMMGPOBSA-N

InChI=1S/C9H10N4O2/c10-8(9(14)15)5-6-1-3-7(4-2-6)12-13-11/h1-4,8H,5,10H2,(H,14,15)/t8-/m0/s1

p-Azido-L-phenylalanine

pAzF; H-4-Azido-Phe-OH; 4-Azido-L-phenylalanine; L-Phe(4-azido)-OH; 4-Azido-L-phenylalanine; 33173-53-4; H-P-AZIDO-PHE-OH; 4-Azidophenylalanine; (S)-2-Amino-3-(4-azidophenyl)propanoic acid; p-azidophenylalanine; p-azido-l-phenylalanine; (2S)-2-amino-3-(4-azidophenyl)propanoic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~25 mg/mL (~121.24 mM)
DMSO : ~5 mg/mL (~24.25 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)