This work was supported by the Medical Research Council, UK. B.O.-S. holds an EMBO fellowship (ATLF 158-2016) and is grateful to H. Pelham and J.W. Chin&#160;for support, and to G. Kym, C. W. Morgan, and O. Perisic for help and advice.

1. Produce and Characterize the Antibody
<ol>
	<li>Express the antibody
	<ol>
		<li>Thaw a vial of HEK suspension cells in a 250&#160;mL flask containing 50 mL of expression medium supplemented with 100 units/mL penicillin, 100 &#181;g/mL streptomycin, and 250 ng/mL amphotericin B. Keep the cells at 37 &#176;C with 8% CO2 in humidified incubators equipped with a shaker at 125 rpm. Split cells to 0.3-0.5 x 106 cells/mL (every 2-3 days) at least 2 times before transfecting.</li>
		<li>When a density of 2.5 x 106 cells/mL is reached (2-3 days after splitting), prepare a fresh solution of 100 mM CypK. For this purpose, weigh 64 mg of CypK, add 2.5 mL of 0.1 sodium hydroxide, vortex, spin down to recover all undissolved particles and sonicate.</li>
		<li>Add 2.5 mL of CypK (100 mM in 0.1 M NaOH) to 42.5 mL of expression medium supplemented with antibiotics. Mix well, add 250 &#181;L of 0.1 M HCl, and sterilize using a 0.22 &#181;m filter.</li>
		<li>Dilute 50 &#181;g of HC and LC pKym1 plasmids21 to 2.5 mL with reduced serum medium. In a separate tube, dilute 135&#160;&#181;L of transfection reagent to 2.5 mL with reduced serum medium.</li>
		<li>Five minutes after preparing the solutions, mix the plasmids and the transfection reagent solution and incubate for 20 min to allow the formation of complexes between the DNA and the transfection reagent.</li>
		<li>In the meantime, centrifuge 125 million cells&#160;at the target density for 5&#160;min at 500 x g, resuspend with the expression medium containing CypK and add the DNA&#8211;transfection reagent mixture. CypK can be purchased or synthesized as reported previously18.</li>
		<li>After incubating cells for 20 h, add 250 &#181;L of transfection reagent enhancers included in the kit.</li>
		<li>Harvest antibodies from the supernatant 6-7 days after addition of CypK (no change of medium is required during expression). 
		*Alternatively, to obtain higher and more consistent yields, a stable cell-line can be generated as described in Oller-Salvia et al. 201821. In this case, trastuzumab(CypK)2 is expressed simply by addition of CypK 5 mM in the expression medium.</li>
	</ol>
	</li>
	<li>Purify the antibody
	<ol>
		<li>Centrifuge the cells for 15 min at 3000 x g.</li>
		<li>Filter the supernatant with a 0.45 or a 0.22 &#181;m filter. If the filter becomes occluded, replace it for a new one and continue filtering. If this happens after only a few milliliters, centrifuge the supernatant for an additional 15 min at 7000 x g.</li>
		<li>Add protein A resin (2 mL/100 mL of supernatant) in an empty polypropylene chromatography column and equilibrate the resin with at least 5 volumes of bead wash buffer (0.1 M sodium phosphate, 150 mM NaCl).</li>
		<li>Split the supernatant into two 50 mL conical tubes and add 5x bead wash buffer (0.5 M sodium phosphate, 150 mM NaCl) followed by the pre-equilibrated protein A resin. Place the conical tube on a roller for 3 h at room temperature to pull down the antibody in the supernatant. 
		Alternatively: Add the supernatant on the column with at least double the recommended volume of resin and allow to flow through. Make sure there is not a significant amount of antibody left in the supernatant by SDS-PAGE. If there is, elute the supernatant through the resin once again.</li>
		<li>Transfer protein A resin with the supernatant into a column and allow the liquid to flow through.</li>
		<li>Add 25&#160;mL of wash buffer (or at least 10 resin volumes)&#160;and allow to flow through.</li>
		<li>Elute the antibody with 4 mL of 0.1 M sodium citrate, pH 3, on 1 mL of 1 M phosphate buffer, 150 mM NaCl.</li>
		<li>Dilute the antibody with 10&#160;mL of PBS, concentrate to 0.5-1&#160;mL by centrifugal filtration, and exchange buffer three times with PBS.</li>
		<li>Purify the samples by FPLC using a butyl HIC column with a flow of 0.5 mL/min and a 0-100% gradient in 30 min of low-salt buffer&#160;(50 mM sodium phosphate pH 7.0, 20% isopropanol) in high-salt buffer (1.5 M (NH4)2SO4, 50 mM sodium phosphate pH 7.0, 5% isopropanol). Collect all fractions and monitor the elution at 280 nm. Small amounts (&#60; 1 mg) can&#160;be purified with HPLC-HIC under the conditions described in 2.4 to maximize yield and purity.</li>
		<li>Pool the fractions that contain antibody, dilute them to at least 4 mL with PBS, concentrate them&#160;by centrifugal filtration and exchange buffer three times with PBS.</li>
	</ol>
	</li>
	<li>Quantify the antibody
	<ol>
		<li>Obtain an approximate concentration by measuring absorbance at 280 nm using a micro-volume spectrophotometer.</li>
		<li>If an accurate measurement is required, use an ELISA kit to measure human IgGs. For a rough estimate, use SDS-PAGE with a Coomassie-based stain as follows:
		<ol>
			<li>Prepare standards by diluting six times two-fold a trastuzumab standard quantified by ELISA at 1 mg/mL.</li>
			<li>Boil the standards and the samples at approximately 0.25 mg/mL in reducing loading buffer.</li>
			<li>Run them in a 4-12% Bis-Tris SDS-PAGE using MES-SDS buffer and stain with a Coomassie-based dye. Finally measure color density of the band corresponding to the light or the heavy chain using ImageJ and interpolate the signal from the samples in the standard curve.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Store the antibody at 4 &#176;C.</li>
</ol>
2. Conjugate the Antibody and Characterize the ADC
<ol>
	<li>Conjugate the antibody with the tetrazine-bearing molecule
	<ol>
		<li>Dilute 10 molar equivalents of tetrazine-vcMMAE (4&#160;&#181;L, 3.4 mM in dimethyl sulfoxide (DMSO)) with 20&#160;&#181;L of acetonitrile and 76&#160;&#181;L of PBS in a small conical tube (e.g.,PCR tube).</li>
		<li>Add 1 molar equivalent of trastuzumab(CypK)2 (100&#160;&#181;L, 2 mg/mL in PBS), mix thoroughly, and allow to react for 3 h at room temperature (25 &#176;C) to form trastuzumab(MMAE)2. 
		Note: Other molecules such as tetrazine-5-TAMRA can be linked to the antibody instead of tetrazine-vcMMAE using this protocol.</li>
	</ol>
	</li>
	<li>Purify the ADC
	<ol>
		<li>Pre-equilibrate a size exclusion spin column with PBS following the manufacturer&#8217;s instructions.</li>
		<li>Add the whole volume of the reaction on the spin column and centrifuge at 1500 x g for 1 min.</li>
	</ol>
	</li>
	<li>Quantify the ADC and analyze the conjugate by SDS-PAGE
	<ol>
		<li>Quantify the ADC as described in 1.3.2. by measuring the color density of the light chain on an SDS-PAGE using the non-modified antibody for the standard curve.</li>
		<li>The heavy chain should have slightly shifted showing an increase in molecular weight upon the conjugation. 
		Note: If the antibody is modified with a fluorophore such as in trastuzumab(TAMRA)2, only the band corresponding to the heavy chain should show in-gel fluorescence before staining.</li>
	</ol>
	</li>
	<li>Analyze the conjugate by HPLC-HIC
	<ol>
		<li>Equilibrate the HPLC-HIC column with 100% buffer A (1.5 M (NH4)2SO4, 50 mM sodium phosphate pH 7.0, 5% isopropanol) for 5 min.</li>
		<li>Mix 15 &#181;L (67 pmol) of a 1 mg/mL solution of trastuzumab(CypK)2 with 15 &#181;L of 2x buffer A in a vial. Then program a 10 &#181;L injection in the HPLC.</li>
		<li>Elute in an isocratic 1 mL/min flow with 100% buffer A for 1 min followed by a 15 min gradient from 100 to 0% of buffer A in B (50 mM sodium phosphate pH 7.0, 20% isopropanol). Monitor the elution at 280 nm.</li>
		<li>Calculate the DAR by integrating the peak corresponding to each species and using the resulting areas in the following equation: 
		DAR = (1x trastuzumab(MMAE) + 2x trastuzumab(MMAE)2) 
		/(trastuzumab(MMAE)0 + trastuzumab(MMAE)1 + trastuzumab(MMAE)2) 
		Expected retention time for trastuzumab(CypK)2 is 7.5-8.0 min, for trastuzumab(CypK, MMAE) is 9.1-9.6 min, and for trastuzumab(MMAE)2 is 10.5-11.0 min.</li>
	</ol>
	</li>
	<li>Analyze the conjugate by LC-MS
	<ol>
		<li>Deglycosylate 30 &#181;L of 1 mg/mL ADC and the unmodified antibody in non-reducing conditions using 250 PNGase F units for at least 6 h at 37 &#176;C.</li>
		<li>Elute the antibody into the mass spectrometer in a C4 1-5 &#181;m 1.0 x 100 mm column using a 20 min gradient of 2% to 80% acetonitrile in water. Acquire data over an m/z range of 350-4000 in positive ion mode with a cone voltage of 150v.</li>
		<li>Deconvolute the raw data using appropriate software. Calculate the difference in mass between the modified and the unmodified antibodies.</li>
	</ol>
	</li>
</ol>
3. Assay Stability of the Dihydropyridazine Linkage in Trastuzumab(TAMRA)2 in Serum
<ol>
	<li>Filter the serum using a 0.22 &#956;m filter under sterile conditions. You can add 100 units/mL penicillin, and 100 &#956;g/mL streptomycin.</li>
	<li>Fill the external wells of a 96-well plate with water. In the central wells, mix in triplicate 90 &#181;L of filtered serum with 10 &#181;L of 1 mg/mL trastuzumab(TAMRA)2 in PBS at a final concentration of 0.1 mg/mL. Place the plate in an incubator saturated with humidity at 37 &#176;C and 4-5% CO2.</li>
	<li>Every 24 h throughout 5 days, pipet each well thoroughly to mix, take a 5 &#181;L aliquot, flash-freeze with liquid nitrogen, and store at -80 &#176;C.</li>
	<li>Once all samples have been collected, thaw the aliquots and analyze using an indirect ELISA as previously reported12 with some modifications:
	<ol>
		<li>Coat a 96-well plate overnight with 0.25 &#181;g/mL HER2 at 4 &#176;C. All other steps are performed at room temperature.</li>
		<li>On the following day, wash 5 times with PBS 0.05% tween (PBS-T) and block with 1% bovine serum albumin for 1 h.</li>
		<li>Wash and incubate the samples at 1:10000 dilution in PBS for 2 h.</li>
		<li>Wash and incubate an anti-TAMRA antibody raised in mouse at 1:2000 dilution in PBS-T with 0.5% BSA for 1 h.</li>
		<li>Wash and incubate an anti-mouse HRP conjugate 1:1000 in PBS-T with 0.5% BSA for 1 h.</li>
		<li>Add TMB and allow to react 5-10 min.</li>
		<li>Stop reaction with 50 &#181;L H2SO4 1 M and measure absorbance at 450 nm. Subtract background measured at 570 nm.</li>
		<li>Fit a 4-parameter logistic regression curve to standard dilutions and interpolate measurements from samples. 
		Note: The stability of trastuzumab(MMAE)2 could be assessed using the same protocol by changing the assay described in 3.3 for a commercial ELISA kit to measure the concentration of the ADC.</li>
	</ol>
	</li>
</ol>
4. Assess the Cytotoxicity of the ADC
Note: This protocol is based on previously reported assays23,26 with some modifications.
<ol>
	<li>Thaw SK-BR-3 and MCF-7 cells and allow them to settle in p25 flasks containing complete medium, i.e. DMEM supplemented with 10% heat inactivated fetal bovine serum, 100 units/mL penicillin, and 100 &#181;g/mL streptomycin.</li>
	<li>Split cells at 80-90% confluence (every&#160;3-4 days) at least 2 times before the assay.</li>
	<li>Two days prior to the assay, fill the outer wells of a 96-well plate with water. Then lift cells with 0.05% trypsin in 0.5 mM EDTA, centrifuge 3 min at 250 x g, resuspend in new medium and seed 3000 cells in 100 &#181;L per (empty) well in the 96-well plates.</li>
	<li>Two days after seeding the cells, prepare 10 serial dilutions of all samples in triplicate with 0.1% DMSO in complete medium. Consider the following samples and controls: trastuzumab(MMAE)2, trastuzumab(CypK)2, trastuzumab wild type, tetrazine-vcMMAE and MMAE.</li>
	<li>Add 100 &#181;L of each sample in each well and incubate for 5 days at 37 &#176;C and 4-5% CO2.</li>
	<li>On day 5, measure cell viability. To this end, use a commercial kit to lyse the cells and measure the ATP released. Plot the percentage of signal with respect to control cells treated with 0.1% DMSO. 
	Note: ADCs can be incubated for 5 days in serum and assayed for cytotoxicity to prove functional stability. 
	CAUTION: MMAE is highly toxic. Therefore use gloves and goggles when handling MMAE derivatives. If you use unmodified MMAE as a control in your experiment, when you prepare the stock solution in DMSO, handle the solid product inside a fume hood.</li>
</ol>

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G., Tona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Lines+as+Candidate+Reference+Materials+for+Quality+Control+of+ERBB2+Amplification+and+Expression+Assays+in+Breast+Cancer.">Cell Lines as Candidate Reference Materials for Quality Control of ERBB2 Amplification and Expression Assays in Breast Cancer.</a> Clinical Chemistry. 55 (7), 1307-1315 (2009).</li><li>Shinmi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-Step+Conjugation+Method+for+Site-Specific+Antibody-Drug+Conjugates+through+Reactive+Cysteine-Engineered+Antibodies.">One-Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive Cysteine-Engineered Antibodies.</a> Bioconjugate Chemistry. 27 (5), 1324-1331 (2016).</li><li>Badescu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+Disulfides+for+Stable+and+Defined+Antibody+Drug+Conjugates.">Bridging Disulfides for Stable and Defined Antibody Drug Conjugates.</a> Bioconjugate Chemistry. 25 (6), 1124-1136 (2014).</li><li>Vazquez-Lombardi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+of+human+antibodies+in+mammalian+cells.">Transient expression of human antibodies in mammalian cells.</a> Nature Protocols. 13 (1), 99-117 (2018).</li><li>Baldi, L., Hacker, D. L., Meerschman, C., Wurm, F. M., Hartley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Expression in Mammalian Cells: Methods and Protocols. , 13-26 (2012).</li></ol>

The authors have nothing to disclose.

The transient expression procedure to produce trastuzumab(CypK)2 described in this protocol is simple and allows for high modularity. The yields obtained are within the ones expected in an academic setting27 and stable cell lines can be generated to further boost the production yield21. During expression, concentrations of CypK lower than 5 mM may result into lower ncAA incorporation, and higher amounts may affect cell growth and decrease antibody yields. CypK as a free amino acid has low water solubility, thus it should be first dissolved at 100 mM in 0.1 M NaOH and then added to the culture medium. After diluting CypK in the medium and before adding it to cells, it is critical to neutralize the medium with HCl and filter to sterilize.&#160;Subsequently, using the transfection reagents specified in this protocol and following the incubation times recommended by the manufacturer is important for a high-yielding expression. For further details on transient expression of human antibodies, the reader is referred to other published protocols31,32.
When the antibody is purified, a high excess of protein A resin is required as indicated to ensure full antibody pull down from the supernatant. In order to prevent the precipitation of trastuzumab during elution, it is recommendable to use a solution with high buffering capacity, dilute immediately with PBS and exchange the buffer avoiding excessive concentration. Always keep the antibody &#60;5&#160;mg/mL.
The conjugation of trastuzumab(CypK)2 with tetrazine-vcMMAE is faster than most reactions reported with other bioorthogonal handles for ADCs. Moreover, this cycladdition occurs under very mild conditions: room temperature or lower and physiological pH. It is important to dilute the DMSO stock solutions of the reactants with acetonitrile prior to addition of PBS and the antibody; otherwise the tetrazine derivatives will precipitate. Acetonitrile is required only due to the high hydrophobicity of MMAE and TAMRA, but less hydrophobic molecules may not need the addition of a co-solvent. Alternatively, tetrazine-vcMMAE can be conjugated without acetonitrile and only 2 molar equivalents of tetrazine-vcMMAE within 20 h. This little amount of toxin could involve a substantial decrease in the manufacturing cost of ADC when compared to current ncAA-based technologies. Trastuzumab(CypK)2 is fully reactive for at least 4 months when preserved at 4 &#176;C.
HPLC-HIC enables an accurate determination of DAR since MMAE is highly hydrophobic and provides an excellent resolution of the peaks corresponding to antibody conjugates with 0, 1 and 2 toxins.&#160;Unreacted tetrazine-vcMMAE elutes around 13.7 min and is detected at 280 nm. This technique requires a starting material with high purity. Moreover, it is not recommendable to quench the reaction with other tetrazine-reactive molecules such as BCN-OH since they can alter the retention times and the shape of the peaks. It is essential that the salt concentration of the samples matches the one in the mobile phase at the start of the gradient in order to obtain a good separation, especially if more than 10-20 &#181;L are injected.
Regarding the LC-MS analysis, deglycosylation of the antibody samples is required to obtain a single peak upon deconvolution of the raw spectrum. The accuracy of the total antibody and ADC masses may vary depending on the callibration of the instrument. Hence, in order to calculate the mass for the modification, subtract the mass obtained for the unmodified antibodies from the one obtained for the ADC. Modern high-resolution mass spectrometers should provide a relative error below 1:10000. Although LC-MS can also be used to calculate the ratio between the different species, this value is usually an overestimation because the modification may affect the ionization capacity of the species generated and low amounts of impurities may not be detected.
The stability of the linker in ADCs is critical because the premature release of the drug results in higher toxicity and lower efficacy; the free cytotoxin damages healthy tissues and the naked antibody competes with the armed one for the target binding sites on diseased cells. A release below 5%, which is within the variability of the stability assay, should be expected.
Finally, the selectivity of an ADC targeting HER2 such as trastuzumab(MMAE)2 can be assessed by comparing the cytotoxicity in SK-BR-3 cells (HER2 high) and MCF-7 cells (HER2 low) since the latter express 15-fold less HER2 receptors than the former28. The immunoconjugate is expected to result into a cell viability at least 2 orders of magnitude lower in SK-BR-3 when compared to MCF-7. The EC50 in SK-BR-3 should be in the two-digit nanomolar range reflecting the high potency of this ADC29,30. The unmodified antibody, either trastuzumab(MMAE)2 or trastuzumab, should show no toxicity in this assay. Tetrazine-vcMMAE should have an effect 3 orders of magnitude lower than the ADC since the linker removes the activity of the peptidomimetic toxin. Conversely, because MMAE is able to permeate the cell membrane30, it should have a similar toxicity to the ADC but display no discrimination between HER2 high and HER2 low cell lines. Moreover, if this assay is performed after a 5-day incubation of the ADC in serum, it can be used to provide a functional proof of the stability of the linker: a release of the toxin would result into either a decrease in ADC efficiency in SK-BR-3 if MMAE was released with part of the linker or a decrease in the selectivity if the linker was cleaved in a traceless fashion.
The ADC technology described herein allows the efficient and site-specific incorporation of a cyclopropene derivative of lysine into IgG1s. Following a facile purification, antibodies can be rapidly conjugated with tetrazine-containing molecules, yielding homogenous products. Due to the small size and high reactivity of the cyclopropene minimal handle, this method should enable the conjugation of sterically hindered payloads. The resulting immunoconjugates are stable in serum and are highly potent and selective. Overall, CypK enables a fast, site-specific and stable bioorthogonal linkage for antibody and other protein conjugates to be used in therapy or diagnosis.

Antibody-drug conjugates (ADCs) combine the selectivity of biotherapeutics and the potency of small cytotoxic molecules. Most ADCs aim to decrease the side effects of traditional chemotherapy by targeting drugs that affect DNA or microtubule polymerization to cancer cells1. First-generation ADCs approved by the Food and Drug Administration (FDA) rely on the modification of lysines and cysteines, which generates mixtures of molecules modified at different positions with decreased pharmacokinetic properties2. By contrast, site-specific conjugation of drugs to the antibodies can generate compounds with improved therapeutic indeces3,4. Seeking to address the challenge of producing homogeneous ADCs, several selective chemical and enzymatic modifications have been reported1,5. However, current methods can target only certain position on the antibody, suffer from low protein expression, provide linkers with low stability, or rely on slow and low-yielding reactions.
Incorporation of non-canonical amino acids (ncAA) through genetic code expansion enables the site-specific installation of a plethora of bioorthogonal reactive groups into proteins, potentially overcoming the limitations of other methods used to generate ADCs. Encoding ncAAs in response to a target (stop) codon relies on aminoacyl-tRNA synthetase/tRNA pairs that are orthogonal to the endogenous pairs that incorporate canonical amino acids6. Several ncAAs have been incorporated into antibodies to generate ADCs. However, most suffer from various liabilities for applications in therapeutic drug conjugation. p-acetylphenylalanine (pAcF)7,8 is not fully bioorthogonal, requires low pH (4.5) and long reaction times (&#62; 60 h), while azides such as p-azidophenylalanine (pAzF)7,9,10, p-azidomethylphenylalanine (pAMF)11, and an azide derivative of lysine (AzK)12,13 may be reduced in the cell14, and the copper used to catalyze Huisgen cycloadditions can induce oxidative damage15.
Although alternative ncAAs based on trans-cyclooctene (TCO), cyclooctyne (SCO) and bicyclo[6.1.0]nonene (BCN) have recently been encoded in an antibody for bio-imaging purposes, the expression system suffers from very low yields (0.5 mg/L)16. Moreover, cyclooctenes and cyclooctynes are large and hydrophobic handles that may increase the susceptibility of the ADC for aggregation -ADC payloads are traditionally hydrophobic and the physicochemical properties of the linker have been shown to greatly impact pharmacokinetics and therapeutic index17. By contrast, 1,3-disubstitued cyclopropenes are small reactive groups that should cause minimal alteration in the protein structure and physichochemical properties18. Cyclopropenes selectively and rapidly react with tetrazines via an inverse electron-demand Diels-Alder cycloaddition19. In this protocol we make use of a derivative of lysine (CypK, Figure 1b) bearing a methyl-cyclopropene that is less affected by steric hindrance than larger strained unsaturated cycles and has a reaction rate constant in the order of 1-30 M-1s-1 in aqueous media18,20.
We recently reported how to incorporate CypK into antibodies to generate ADCs by reacting this minimal bioorthogonal handle with tetrazine-bearing molecules21. Here we describe the ADC preparation procedure in more detail with emphasis on the most challenging steps. The incorporation of CypK is directed using a pyrrolysyl-tRNA synthetase(PylRS)/tRNACUA(PylT) pair in response to an amber codon introduced in the antibody heavy chain (HC)22. Here we use two plasmids for transient transfection (Figure 1a), one encoding the heavy chain of the antibody and the other one encoding the light chain (LC), both containing the PylRS/PylT cassette. Alternatively, a stable cell line that enables higher antibody yields can be generated through a more laborious procedure21.
The aforementioned expression systems can produce the therapeutic anti-HER2 immunoglobulin 1 (IgG1) trastuzumab with CypK at similar levels to the wild type antibody. We selected the first position of the CH1 domain on the heavy chain to encode the ncAA (HC-118TAG). This is the most commonly modified site in ADCs23 and is known as HC-118 (EU numbering) but has also been referred to as HC-121 (sequence position) and HC-114 (Kabat numbering)24. Since this position is conserved throughout all IgG1s, these expression systems should be amenable to most therapeutic antibodies.
We show trastuzumab(CypK)2 can be easily purified by protein A followed by fast protein liquid chromatography with a hydrophobic interaction column (FPLC-HIC). Subsequently the antibody is covalently linked within 3 h to the microtubule polymerization inhibitor monomethyl auristatin E (MMAE), which is used in the FDA-approved ADC Adcetris. Here we use a benzyl-tetrazine derivative of MMAE (tetrazine-vcMMAE) with a linker comprising a glutarate spacer and a valine-citrulline protease-labile component followed by a p-aminobenzylalcohol self-immolative unit; this linker is cleaved by Cathepsin B in the lysosome upon internalization of the ADC resulting in the traceless release of the toxin25. In order to show the broad scope of the reaction, the antibody is also linked to the fluorophore tetramethylrhodamine (TAMRA). We explain how to verify the identity of the conjugate by liquid chromatography coupled to mass spectrometry (LC-MS) and to calculate the drug-to-antibody ratio (DAR) using high performance liquid chromatography with a hydrophobic interaction column (HPLC-HIC).
As part of the characterization of the antibody performance, we describe how to test the stability of the dihydropyridazine linkage in human serum. This parameter is more easily assessed in trastuzumab-TAMRA because it can be quantified by a simple ELISA and the interpretation of the results is not complicated by the protease labile component of trastuzumab(MMAE)2. Finally, the selectivity and potency of trastuzumab(MMAE)2 is assessed by comparing the cytoxicity of the ADC across cell lines expressing different levels of HER2. This assay also provides a functional proof of the ADC stability when performed after incubating the immunoconjugate in human serum.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Expi293F</td>
			<td >ThermoFisher Scientific</td>
			<td >A14527</td>
			<td >HEK suspension cells</td>
		</tr>
		<tr >
			<td >Expi293 Expression Medium</td>
			<td >ThermoFisher Scientific</td>
			<td >A1435101</td>
			<td>Expression medium</td>
		</tr>
		<tr >
			<td >Antibiotic-antimycotic</td>
			<td >ThermoFisher Scientific</td>
			<td >15240062</td>
			<td >Penicillin-streptomycin-amphotericin B</td>
		</tr>
		<tr >
			<td >125mL Polycarbonate Erlenmeyer Flask with Vent Cap</td>
			<td >Corning</td>
			<td >431143</td>
			<td>Shake flasks</td>
		</tr>
		<tr >
			<td >Brunswick S41i incubator</td>
			<td >Eppendorf</td>
			<td >S41I230011</td>
			<td>CO2 incubator with a shaker</td>
		</tr>
		<tr >
			<td >Sodium hydroxide 4 mol/l (4 N) in aqueous solution</td>
			<td >VWR</td>
			<td >191373M</td>
			<td></td>
		</tr>
		<tr >
			<td >Cyclopropene lysine</td>
			<td >Sichem</td>
			<td >SC-8017</td>
			<td>In this study it was synthesized as described by Elliot et al. 2014</td>
		</tr>
		<tr >
			<td >Steriflip-GP, 0.22 &#181;m, polyethersulfone, gamma irradiated</td>
			<td >Merck Millipore</td>
			<td >SCGP00525</td>
			<td></td>
		</tr>
		<tr >
			<td >Opti-MEM, Reduced Serum Medium</td>
			<td >ThermoFisher Scientific</td>
			<td >31985070</td>
			<td>Reduced serum medium</td>
		</tr>
		<tr >
			<td >ExpiFectamine 293 Transfection Kit</td>
			<td >ThermoFisher Scientific</td>
			<td >A14525</td>
			<td>Transfection reagent</td>
		</tr>
		<tr >
			<td >5810 R centrifuge</td>
			<td >Eppendorf</td>
			<td >5811000460</td>
			<td></td>
		</tr>
		<tr >
			<td >Millex-GP Syringe Filter Unit, 0.22 &#181;m, polyethersulfone, 33 mm, gamma sterilized</td>
			<td >Merck Millipore</td>
			<td >SLGP033RS</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein A resin</td>
			<td >Sino Biological</td>
			<td >10600-P07E-RN-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Poly-Prep Chromatography Columns, Pkg of 50</td>
			<td >Bio-Rad</td>
			<td >7311550</td>
			<td>Polypropylene chromatography column</td>
		</tr>
		<tr >
			<td >Econo-Column Funnel</td>
			<td >Bio-Rad</td>
			<td >7310003</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium citrate</td>
			<td >Fluka</td>
			<td >71635</td>
			<td></td>
		</tr>
		<tr >
			<td >&#196;KTA explorer FPLC</td>
			<td >GE Healthcare</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >HiTrap HIC Selection Kit</td>
			<td >GE Healthcare</td>
			<td >28-4110-07</td>
			<td >Includes HiTrap 1 mL Butyl HP</td>
		</tr>
		<tr >
			<td >Ammonium sulfate</td>
			<td >VWR</td>
			<td >2133.296</td>
			<td></td>
		</tr>
		<tr >
			<td >Isopropanol</td>
			<td >Honywell</td>
			<td >34863-2.5L</td>
			<td></td>
		</tr>
		<tr >
			<td >Dymethyl sulfoxide</td>
			<td >Sigma-Aldrich</td>
			<td >D8418-50ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Tetrazine-vcMMAE</td>
			<td >ChemPartner</td>
			<td >-</td>
			<td>Costum synthesized</td>
		</tr>
		<tr >
			<td >Tetrazine-5-TAMRA</td>
			<td >Jena Bioscience</td>
			<td >CLK-017-05</td>
			<td></td>
		</tr>
		<tr >
			<td >NuPAGE 4-12% Bis-Tris Gel 1.0mm x 10 well</td>
			<td >ThermoFisherScientific</td>
			<td >NP0321BOX</td>
			<td></td>
		</tr>
		<tr >
			<td >Xcell SureLock Mini-Cell</td>
			<td >ThermoFisherScientific</td>
			<td >EI0001</td>
			<td></td>
		</tr>
		<tr >
			<td >UltiMate 3000 HPLC</td>
			<td >ThermoFisherScientific</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Scientific MAbPac, HIC-20, 4.6 x 100 mm, 5 &#181;m</td>
			<td >ThermoFisherScientific</td>
			<td >088553</td>
			<td></td>
		</tr>
		<tr >
			<td >PNGase F</td>
			<td >New England BioLabs</td>
			<td >P0704S</td>
			<td></td>
		</tr>
		<tr >
			<td >NanoAcquity</td>
			<td >Waters</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >C4 BEH 1-5 &#181;m 1.0 x 100 mm UPLC column&#160;</td>
			<td >Waters</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >96-well microplates for cell culture</td>
			<td >ThermoFisherScientific</td>
			<td >156545</td>
			<td></td>
		</tr>
		<tr >
			<td >Human serum</td>
			<td >Sigma-Aldrich</td>
			<td >H4522-20mL</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 incubator</td>
			<td >Panasonic</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >HER2 ECD</td>
			<td >Sino Biological</td>
			<td >10004-HCCH</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-TAMRA</td>
			<td >Abcam</td>
			<td >an171120</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-mouse HRP</td>
			<td >Santa Cruz</td>
			<td >sc-2005</td>
			<td></td>
		</tr>
		<tr >
			<td >TMB</td>
			<td >BioLengend</td>
			<td >421101</td>
			<td></td>
		</tr>
		<tr >
			<td >Sulfuric acid</td>
			<td >Sigma-Aldrich</td>
			<td >84727-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >PHERAstar FS</td>
			<td >BMG Labtech</td>
			<td ></td>
			<td>Plate reader</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Sigma-Aldrich</td>
			<td >D5671-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >SK-BR-3</td>
			<td >ATCC</td>
			<td >HTB-30</td>
			<td></td>
		</tr>
		<tr >
			<td >MCF-7</td>
			<td >ATCC</td>
			<td >HTB-22</td>
			<td></td>
		</tr>
		<tr >
			<td >CellTiterGlo 2.0 Assay</td>
			<td >Promega</td>
			<td >G9242</td>
			<td>Cell viability assay based on the measurement of ATP released after cell lysis. The output signal is luminscence.</td>
		</tr>
		<tr >
			<td >Monomethyl Auristatin E</td>
			<td >Cayman Chemical</td>
			<td >16267</td>
			<td></td>
		</tr>
		<tr >
			<td >NanoDrop 2000</td>
			<td >ThermoFisherScientific</td>
			<td ></td>
			<td>Microvolume spectrophotometer</td>
		</tr>
		<tr >
			<td >Human IgG ELISA Quantificaiton Set</td>
			<td >Bethyl</td>
			<td >E80-104</td>
			<td></td>
		</tr>
		<tr >
			<td >MaxEnt1 in MassLynx</td>
			<td >Waters</td>
			<td ></td>
			<td>Software application for mass spectrum deconvolution</td>
		</tr>
		<tr >
			<td >IntantBlue</td>
			<td >Expedeon</td>
			<td >ISB1L</td>
			<td>Coomassie-based stain</td>
		</tr>
		<tr >
			<td >Intact MMAE-ADC ELISA Kit (Sandwich Assay)</td>
			<td >Epitope Diagnostics, Inc.</td>
			<td >KTR 782</td>
			<td></td>
		</tr>
		<tr >
			<td >Tube 50 mL, 114x28mm, PP</td>
			<td >Sarstedt</td>
			<td >62.547.254</td>
			<td>Conical tube</td>
		</tr>
		<tr >
			<td >Amicon Ultra-15 Centrifugal Filter Units 50,000 NMWL</td>
			<td >Merck</td>
			<td >UFC905024</td>
			<td>Centrifugal filtration concentrator (after protein A pull down)</td>
		</tr>
		<tr >
			<td >Amicon Ultra-4 Centrifugal Filter Units 50,000 NMWL</td>
			<td >Merck</td>
			<td >UFC805024</td>
			<td>Centrifugal filtration concentrators (after FPLC or HPLC purification)</td>
		</tr>
		<tr >
			<td >Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td >ThermoFisherScientific</td>
			<td >89882</td>
			<td>Size exclusion spin columns</td>
		</tr>
		<tr >
			<td >Tube PCR 0.2ml Flat Cap</td>
			<td >Thistle Scientific Ltd</td>
			<td >AX-PCR-02-C-CS</td>
			<td>PCR tubes</td>
		</tr>
		<tr >
			<td >Nunc MaxiSorp&#170; flat-bottom</td>
			<td >ThermoFisherScientific</td>
			<td >44-2404-21</td>
			<td>Plates for ELISA</td>
		</tr>
	</tbody>
</table>

genetic encoding of a non-canonical amino acid for the generation of antibody-drug conjugates through a fast bioorthogonal reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

The reported transient expression system (Figure 1a) yields 22 &#177; 2 mg of trastuzumab(CypK)2 per liter of culture medium, which represents 2/3 of the wild type antibody produced under the same conditions (Figure 1c). The stable cell line can increase this yield up to 31 &#177; 2 mg/L21.
Trastuzumab(CypK)2 can be conjugated with tetrazine-vcMMAE, which yields quasi homogeneous trastuzumab(MMAE)2 within 3 h at 25 &#176;C (Figure 2). The high hydrophobicity of this cytotoxin requires addition of 10% acetonitrile when 5 or more molar equivalents of toxin per CypK are used. Alternatively, the cycloaddition is also completed within 20 h using 2 equivalents of tetrazine-vcMMAE without acetonitrile (Figure 2c). Trastuzumab(CypK)2 reacts with tetrazine-TAMRA within 2 h at 25 &#176;C and 3-6 h are required when the temperature is decreased to 4 &#176;C (Figure 3c).
The expected DAR for trastuzumab(MMAE)2 measured by HPLC-HIC is 1.9 (Figure 2b). The peak initially observed in the chromatogram at 8.0 min represents the unconjugated antibody (DAR 0) and should have completely disappeared when the reaction is completed. The species with DAR 1 elutes at 9.1-9.6 min and should have an area &#60; 10% after 3 h; and the target product with DAR 2 has a retention time of 10.5-11.0 with an expected area &#62; 90%. The mobility shift and fluorescence in SDS-PAGE gels confirms the incorporation of TAMRA (Figure 3b) and the identity of the immunoconjugates is verified by LC-MS (Figure 2d-e and Figure 3d).
Incubation of trastuzumab(TAMRA)2 for 5 days in human serum and subsequent analysis by ELISA confirms that the payload remains attached to the antibody (Figure 4b). Regarding the cytotoxicity assay, trastuzumab(MMAE)2 shows high potency in SK-BR-3 (HER2 high) breast cancer cells, with a half maximal effective concentration (EC50) of 55 &#177; 10 pM (Figure 4d). Trastuzumab(MMAE)2 maintains the cytotoxicity after 5 days of incubation in human serum (Figure 4c). Conversely, when the ADC is assayed on MCF-7 (HER2 low) the EC50 is 200-fold lower (Figure 4d). The wild type antibody, trastuzumab(CypK)2 and tetrazine-vcMMAE show extremely low toxicity (Figures 4d and 4e), whereas MMAE displays high non-selective cytotoxicity in both cell lines (Figure 4e).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58066/58066fig1.jpg" /> 
Figure 1: Transient expression system. A. Relevant regions of the plasmids used for transient transfection in HEK293 cells. CMV: cytomegalovirus promoter, WPRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element, PylT: pyrrolysyl tRNA, U6: specific promoter, PylRS: pyrrolysil tRNA synthetase, &#62; and &#60;: direction of transcription. B. N&#949;-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-L-lysine (CypK). C. Expression yields of wild type (WT) trastuzumab and trastuzumab(CypK)2 as measured in a western blot after protein A purification. Error bars represent the standard deviation of biological triplicates. This figure has been modified with permission from Oller-Salvia et al. 201821. <a href="https://www.jove.com/files/ftp_upload/58066/58066fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58066/58066fig2.jpg" /> 
Figure 2: Conjugation of trastuzumab(CypK)2 with tetrazine-vcMMAE. A. Inverse electron-demand Diels-Alder cycloaddition between the CypK residue in the antibody and the tetrazine derivative of MMAE. The reacting groups are highlighted in red, p-aminobenzylalcohol is depicted in green and the valine-citrulline dipeptide in blue. B. HPLC-HIC chromatograms showing the progress of the conjugation of antibody. C. Degree of conversion with respect to the maximum DAR of 1.9 using different reagent concentrations. D-E. Deconvoluted mass spectra of the full-length antibody before and after the conjugation. Trastuzumab(CypK)2 was obtained using the stable cell line. This figure has been modified with permission from Oller-Salvia et al. 201821. <a href="https://www.jove.com/files/ftp_upload/58066/58066fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58066/58066fig3.jpg" /> 
Figure 3: Conjugation of trastuzumab(CypK)2 with tetrazine-TAMRA. A. Inverse electron-demand Diels-Alder cycloaddition between the CypK residue in the antibody and the tetrazine derivative of TAMRA. B. SDS-PAGE gels showing the mobility shift and the in-gel fluorescence originated by the conjugation of TAMRA. C. Conjugation kinetics at two different temperatures. D. Deconvoluted mass spectrum of trastuzumab(TAMRA)2. Trastuzumab(CypK)2 was obtained using the stable cell line. This figure has been modified with permission from Oller-Salvia et al. 201821. <a href="https://www.jove.com/files/ftp_upload/58066/58066fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58066/58066fig4.jpg" /> 
Figure 4: Stability in serum and cytotoxicity of trastuzumab conjugates. A. Cartoon highlighting the features desired in an internalizing ADC. B. Stability of trastuzumab(TAMRA)2 in human serum as measured in ELISA. C. Cell viability assay with trastuzumab(MMAE)2 after 5 days in human serum (+ serum, black). A control sample incubated in PBS instead of serum (- serum, red) was included in the same assay. D. Cell viability assay with freshly diluted antibody samples. E. Cell viability assay with freshly diluted MMAE derivatives. Error bars represent the standard error of the mean of 3 independent experiments. This figure has been modified with permission from Oller-Salvia et al. 201821. <a href="https://www.jove.com/files/ftp_upload/58066/58066fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction at JoVE.com

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

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Chemistry

7.6K Views.  Medical Research Council Laboratory of Molecular Biology. Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Video: Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

Temperature-programmed Deoxygenation of Acetic Acid on Molybdenum Carbide Catalysts

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system includes a reactor, furnace, gas and vapor sources, flow control, instrumentation to quantify reaction products (e.g., gas chromatograph), and instrumentation to monitor the reaction in real time (e.g., mass spectrometer). Here, we apply the TPRxn methodology to study molybdenum carbide catalysts for the deoxygenation of acetic acid, an important reaction among many in the upgrading/stabilization of biomass pyrolysis vapors. TPRxn is used to evaluate catalyst activity and selectivity and to test hypothetical reaction pathways (e.g., decarbonylation, ketonization, and hydrogenation). The results of the TPRxn study of acetic acid deoxygenation show that molybdenum carbide is an active catalyst for this reaction at temperatures above ca. 300 &#176;C and that the reaction favors deoxygenation (i.e., C-O bond-breaking) products at temperatures below ca. 400 &#176;C and decarbonylation (i.e., C-C bond-breaking) products at temperatures above ca. 400 &#176;C.

Presented here is a protocol for the operation of a micro-scale temperature-programmed reactor for evaluating the catalytic performance of molybdenum carbide during acetic acid deoxygenation.

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...