Cyclopropene amino acids and methods
10774039 ยท 2020-09-15
Assignee
Inventors
Cpc classification
C07C271/22
CHEMISTRY; METALLURGY
C07K1/1072
CHEMISTRY; METALLURGY
C07C269/06
CHEMISTRY; METALLURGY
C12Y601/01
CHEMISTRY; METALLURGY
International classification
C07K14/00
CHEMISTRY; METALLURGY
C07C271/22
CHEMISTRY; METALLURGY
C07C269/06
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
Abstract
The invention relates to a polypeptide comprising an amino acid having a cyclopropene group wherein said cyclopropene group is joined to the amino acid via a carbamate group. Suitably the cyclopropene group is a 1,3-disubstituted cyclopropene such as a 1,3-dimethylcyclopropene. Suitably the cyclopropene group is present as a residue of a lysine amino acid. The invention also relates to methods of making the polypeptides. The invention also relates to an amino acid comprising cyclopropene wherein said cyclopropene group is joined to the amino acid moiety via a carbamate group.
Claims
1. A polypeptide comprising an amino acid having a cyclopropene group wherein said cyclopropene group is linked to the amino acid via a carbamate group and the carbamate group does not form a part of the polypeptide backbone, wherein said cyclopropene group is a 1,3-disubstituted cyclopropene.
2. A polypeptide according to claim 1 wherein said cyclopropene is a 1,3-dimethylcyclopropene.
3. A polypeptide according to claim 1 wherein said cyclopropene group is present as a residue of a lysine amino acid.
4. A polypeptide according to claim 1 further comprising a tetrazine compound linked to said cyclopropene group.
5. An amino acid comprising cyclopropene wherein said cyclopropene group is linked to the amino acid moiety via a carbamate group and the carbamate group does not form a part of the peptide backbone, wherein said cyclopropene group is a 1,3-disubstituted cyclopropene.
6. An amino acid according to claim 5 wherein said cyclopropene is a 1,3-dimethylcyclopropene.
7. An amino acid according to claim 5 wherein said amino acid is a lysine amino acid.
8. An amino acid according to claim 7 which comprises N.sup.-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-1-lysine.
9. An amino acid according to claim 7 which consists of ##STR00006##
10. A method of producing a polypeptide comprising a cyclopropene group wherein said cyclopropene group is joined to an amino acid moiety of the polypeptide via a carbamate group, said method comprising genetically incorporating said amino acid comprising said cyclopropene group joined to said amino acid moiety via said carbamate group, into said polypeptide.
11. A method according to claim 10 wherein producing the polypeptide comprises (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a cyclopropene group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said amino acid having a cyclopropene group into the polypeptide chain.
12. A method according to claim 10 wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MbtRNA.sub.CUA and said tRNA synthetase comprises MbPylRS; or wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises MmtRNA.sub.CUA and said tRNA synthetase comprises MmPylRS.
13. A method according to claim 10 wherein said carbamate group does not form a part of the peptide backbone, and wherein said cyclopropene group is a 1,3-disubstituted cyclopropene.
14. A method of producing a polypeptide comprising a tetrazine group, said method comprising providing a polypeptide according to claim 1, contacting said polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine to a cyclopropene group of the polypeptide by an inverse electron demand Diels-Alder cycloaddition reaction.
15. A method according to claim 14 wherein said reaction is allowed to proceed for 10 minutes or less, preferably for 1 minute or less, preferably for 30 seconds or less.
16. A polypeptide according to claim 1 wherein said polypeptide comprises two or more amino acids each having a cyclopropene group, wherein each said cyclopropene group is linked to each said amino acid via a carbamate group and wherein each carbamate group does not form a part of the polypeptide backbone.
17. A polypeptide according to claim 16 wherein said polypeptide comprises four amino acids each having a cyclopropene group.
18. An antibody drug conjugate (ADC) comprising a polypeptide according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
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(11) T4 lysozyme isolated after SORT with UUU(Lys) in the presence of 1 mM 3. Expected mass WT T4 lysozyme: 19512.2 Da, Found mass: 195102.0 Da. Expected mass WT T4 lysozyme Lys 3.fwdarw.single mutation: 19622.3 Da, Found mass: 196202.0 Da.
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(13) Chemically competent DH10B cells were transformed with two plasmids: pBKwtPylRS necessary for expression of PylRS, and pBAD_wtT4L_MbPylT.sub.XXX plasmids that is required for expression of PyltRNA.sub.XXX and expresses lysozyme under arabinose control. The cells were recovered in 1 ml SOB medium for one hour at 37 C. prior to aliquoting to 10 ml LB-KT (LB media with 50 g ml.sup.1 kanamycin, and 25 g ml.sup.1 tetracycline) and incubated overnight (37 C., 250 rpm, 12 h). The overnight culture (OD.sub.6003) was diluted to a OD.sub.6000.3 in 10 mL LB-KT.sub.1/2 (LB media with 25 g ml.sup.1 kanamycin, and 12.5 g ml.sup.1 tetracycline) supplemented with 3 at different concentrations, 0, 0.1, 0.5 mM. 200 L aliquots of these cultures were transferred into a 96-well plate and OD.sub.600 measured using a Microplate reader, Infinite 200 Pro (TECAN). OD.sub.600 was measured for each sample every 10 min with linear 1 mm shaking between the measurements.
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(15) Chemically competent DH10B cells were transformed with two plasmids: pBKwtPylRS necessary for expression of PylRS, and pBAD_wtT4LMbPylT.sub.UUU plasmid that is required for expression of PyltRNA.sub.UUU. pBAD_wtT4L_MbPylT.sub.UUU plasmid also contains the gene for expression of T4 lysozyme that is downstream of arabinose-inducible promoter. After transformation, cells were recovered in 1 ml SOB medium for one hour at 37 C. prior to inoculation in 10 ml LB-KT (LB media with 50 g ml.sup.1 kanamycin, and 25 g tetracycline). The culture was incubated overnight (37 C., 250 rpm, 12 h) and subsequently diluted to an OD.sub.6000.3 in 30 mL LB-KT.sub.1/2 (LB media with 25 g ml.sup.1 kanamycin, and 12.5 g ml.sup.1 tetracycline) supplemented with 3 at different concentrations, 0, 0.1, 0.5 mM. The cultures was incubated (37 C., 250 rpm) for 1 h, when OD.sub.600 reached approximately 0.6. 2 ml culture aliquot was collected in a separate tube for each of three cultures. This is the pre-induction culture (lane labelled as 1 in the gel image). Subsequently arabinose was added at a final concentration of 0.2% (v/v) to induce expression of T4 lysozyme and culture aliquots of 2 mL were collected every hour (lanes labelled as 2, 3 and 4 corresponding to 1, 2 and 3 h culture collection after induction). For each of the collected cultures, bacterial cells were pelleted by centrifugation at 4 C., washed with ice cold PBS (31 mL) and subsequently the pellets were frozen and stored at 20 C. The pellets were then thawed in 200 L of ice cold PBS and lysed by sonication (910 s ON/20 s OFF, 70% power). The lysates were clarified by centrifugation at 15,000 RPM, 4 C. for 30 minutes. The supernatants were transferred to fresh 1.5 mL tubes. 50 L of supernatant was transferred to a new tube for the labeling reactions, and the rest was frozen in liquid nitrogen and stored at 80 C. To the 50 L of supernatant, 0.5 L of 2 mM 4a was added and the lysates were incubated at 25 C. for 1 hour. After 1 h, 17 L of 4LDS sample buffer supplemented (6 mM BCN and 5% BME) was added and mixed by vortexing gently. Samples were incubated for 10 min before boiling at 90 C. for 10 min. Samples were analysed by 4-12% SDS-PAGE and fluorescent images were acquired using Typhoon Trio phosphoimager (GE Life Sciences)
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(25) GFP (amino acid residues 1-238), Amber codon at position 248, mCherry (amino acid residues 255-489), HA tag (amino acid residues 491-499), Myc tag (amino acid residues 500-509), His tag (amino acid residues 510-515) and SV40 NLS (amino acid residues 523-528).
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EXAMPLESDESCRIPTION OF THE EMBODIMENTS
(27) Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
Chemical SynthesesGeneral Methods
(28) All chemicals and solvents were purchased from Sigma-Alrich, Alfa Aesar or Fisher Scientific and used without further purification unless otherwise stated. Qualitative analysis by thin layer chromatography (TLC) was performed on aluminium sheets coated with silica (Merck TLC 60F-254). The spots were visualized under short wavelength ultra-violet lamp (254 nm) or stained with basic, aqueous potassium permanganate, ethanolic ninhydrin or vanillin. Flash column chromatography was performed with specified solvent systems on silica gel 60 (mesh 230-400).
(29) LC-MS analysis was performed on Agilent 1200 machine. The solvents used consisted of 0.2% formic acid in water (buffer A) and 0.2% formic acid in acetonitrile (buffer B). LC was performed using Phenomenex Jupiter C18 column (1502 mm, 5 m) and monitored using variable wavelengths. Retention times (R.sub.t) are recorded to a nearest 0.1 min and m/z ratio to nearest 0.01 mass units. The following programme was used for small molecule LC gradient: 0-1 min (A:B 10:90-10:90, 0.3 mL/min), 1-8 min (A:B 10:90-90:10, 0.3 mL/min), 8-10 min (A:B 90:10-90:10, 0.3 mL/min), 10-12 (A:B 90:10-10:90, 0.3 mL/min).
(30) Mass spectrometry analysis following LC was carried out in ESI mode on a 6130 Quadrupole spectrometer and recorded in both positive and negative ion modes. NMR analysis was carried out on a Bruker 400 MHz instrument. All reported chemical shifts () relative to TMS were referenced to the residual protons in deuterated solvents used: d.sub.1chloroform (.sup.1H =7.26 ppm, .sup.13C =77.16 ppm), d.sub.6dimethylsulfoxide (.sup.1H =2.49 ppm, .sup.13C =39.52 ppm), D.sub.2O (.sup.1H =4.70). APT or two-dimensional experiments (COSY, HSQC) were always performed to provide additional information used for analysis where needed. Coupling constants are given in Hz and described as: singlets, doubletd, triplett, quartetq, broad singletbr, multipletm, doublet of doubletsdd, etc. and combinations thereof.
Protein Expression, Purification and Labelling of Site-Specifically Incorporated 3 in E. coli
(31) Expression and purification of sfGFP-3 from E. coli Electrocompetent E. coli DH10B cells were co-transformed with pBK-MbPylRS and psfGFP150TAG PyIT.sup.14, 26. Transformed cells were recovered in S.O.B. (1 mL, supplemented with 0.2% glucose) for 1 h at 37 C. and used to inoculate LB containing 50 g/mL kanamycin and 25 g/mL tetracycline (LB-KT). The cells were incubated with shaking overnight at 37 C., 250 r.p.m. 1 mL of overnight culture was used to inoculate 100 mL of LB-KT, the day culture was then incubated (37 C., 250 r.p.m). At O.D..sub.6000.3, the culture was divided equally and supplemented with either 3 (1 mM) or H.sub.2O (500 L) and incubated further (37 C., 250 r.p.m). At O.D..sub.6000.6 protein expression was induced by the addition of arabinose (0.2%), after 4 h, the cells were harvested by centrifugation (4000 r.p.m, 20 min) and the pellet frozen until further use.
(32) The frozen bacterial pellet was thawed on ice and resuspended in 2.5 mL lysis buffer (Bugbuster, Novagen, 50 g/mL DNAse 1, Roche inhibitor cocktail and 20 mM imidazole). Cells were incubated (4 C., 30 minutes) then clarified by centrifugation (16000 g, 4 C., 30 minutes). The clarified lysates were transferred to fresh tubes and 100 L Ni-NTA slurry added. The mixtures was incubated with agitation (4 C., 1 h) and then collected by centrifugation (1000 g, 4 C., 5 min). The beads were resuspended three times in 500 L wash buffer (10 mM Tris-HCL, 40 mM imidazole, 200 mM NaCl, pH 8) and collected by centrifugation (1000 g, 4 C., 5 min). Finally, the beads were resuspended in 100 L, elution buffer (10 mM Tris-HCL, 300 mM imidazole, 200 mM NaCl, pH 8), pelleted by centrifugation (1000 g, 4 C., 5 min) and the supernatant collected into fresh tubes. The elution was repeated three times with 100 L of elution buffer. The purified proteins were analysed by 4-12% SDS-PAGE and LC-MS.
Protein Mass Spectrometry
(33) Using an Agilent 1200 LC-MS system, ESI-MS was additionally carried out with a 6130 Quadrupole spectrometer. The solvent system consisted of 0.1% formic acid in H.sub.2O as buffer A, and 0.1% formic acid in acetonitrile (MeCN) as buffer B. Protein UV absorbance was monitored at 214 and 280 nm. Protein MS acquisition was carried out in positive ion mode and total protein masses were calculated by deconvolution within the MS Chemstation software (Agilent Technologies).
In Vitro Labeling of Purified sfGFP150-3
(34) To Purified sfGFP150-1 or sfGFP150-3 protein (30 M, in elution buffer) was added 4a (10 molar equivalents, from a 2 mM stock solution in DMSO). The reactants were mixed by aspirating several times and the mixture then incubated at room temperature for 2 hours, a sample was analysed by ESI-MS. Following incubation the proteins were separated by 4-12% SDS-PAGE and analysed by using Typhoon Trio phosphoimager (GE Life Sciences).
Time Course of sfGFP150-3 and sfGFP150-NorK Labelling and Rate Constant Determination
(35) 2 nmol sfGFP-3 (10.6 M) was labeled at room temperature by the addition of 20 nmol of tetrazine-dye conjugate 4a (10 l of a 2 mM solution in DMSO) the samples were mixed by aspirating several times. At different time points, 8 L aliquots were taken from the solution and quenched with a 700-fold excess of bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and plunged into liquid nitrogen. Samples were mixed with NuPAGE LDS sample buffer supplemented with 5% -mercaptoethanol, heated for 10 min to 90 C. and analyzed by 4-12% SDS page. The amounts of labelled proteins were quantified by scanning the fluorescent bands with a Typhoon Trio phosphoimager (GE Life Sciences). Bands were quantified with the ImageQuant TL software (GE Life Sciences) using rubber band background subtraction. The rate constant was determined by fitting the data to a single-exponential equation. The calculated observed rate k was divided by the concentration of 4a to obtain rate constant k for the reaction. Measurements were done in triplicate. All data processing was performed using Kaleidagraph software (Synergy Software, Reading, UK). For comparison the rate of labelling sfGFP bearing Ne-5-norbornene-2-yloxycarbonyl-L-lysine (NorK), a known substrate for PylRS, was determined in a similar way using 11.25 mM sfGFP bearing NorK at position 150 (SfGFP-NorK) and 20 equivalents of 4a.
Plasmid Construction for pBAD_wtT4L_MbPylT.SUB.XXX
(36) pBAD_T4L83TAG_MbPylT.sub.CUA was digested with Ncol and Kpnl restriction enzymes. The same restriction enzymes were also used to digest the wild-type T4 lysozyme from (D67) pBAD_wtT4L. The insert and backbone were ligated in 3:1 ratio using T4 DNA ligase (RT, 2 hours), transformed into chemically competent DH10B cells and grown on Tetracycline agar plates (37 C., 18 hours). Single colonies were picked and the correct sequence was confirmed by DNA sequencing (GATC Gmbh.), this step created pBAD_wtT4L_MbPylT.sub.CUA. All final constructs were confirmed by DNA sequencing.
Proteomic Incorporation of 3 Via SORT in E. coli Expressing T4 Lysozyme
(37) Electrocompetent E. coli DH10B cells (50 L) were either doubly transformed with pBAD_wtT4L_MbPylT.sub.XXX plasmid (2 L, necessary for expression of PyltRNA.sub.XXX and expresses T4 lysozyme under arabinose control) and pBKwtPylS plasmid (2 L necessary for expression of PylRS) or singly transformed with pBAD_wtT4L_MbPylT.sub.XXX alone. Transformed cells were recovered in 1 mL S.O.B. (supplemented with 0.2% glucose) for 1 h at 37 C. 100 L of the recovery was used to inoculate 5 mL LB-KT (50 g/mL kanamycin and 25 g/mL tetracycline) or LB-T (25 g/mL tetracycline). Cultures were incubated overnight (37 C., 250 r.p.m.). 1 mL of each overnight culture was used to inoculate 15 mL strength antibiotic containing media LB-T or LB-KT. Cultures were incubated at 37 C. until O.D..sub.6000.3 was reached, at this time each culture was divided into 5 mL aliquots and supplemented with either 3 (0.1 mM final conc.) or H.sub.2O (50 L). Cultures were then incubated (37 C., 250 r.p.m.). At O.D..sub.600 0.6. T4 lysozyme expression was initiated by the addition of arabinose (0.2% final conc.) and cultures incubated for a further 4 hours. Cells were harvested by centrifugation (4000 rpm, 4 C., 20 minutes) and then resuspended three times in 1 mL of ice cold PBS and collected by centrifugation (4000 rpm, 4 C., 20 minutes). The final bacterial pellets were immediately frozen for storage.
E. coli: Chemoselective Labelling Proteomes Tagged with 3 with Tetrazine-Dye Conjugates
(38) Frozen bacterial pellets were resuspended in 500 L PBS and lysed using a bath sonicator (energy output 7.0, 90 s total sonication time. 10 s blasts and 20 s breaks, Misonix Sonicator 3000). The lysate was cleared by centrifugation (4 C., 14000 r.p.m., 30 min) and the supernatant aspirated to a fresh tube. To 50 L of cleared cell lysate was added. 4a (2 mM, stock in DMSO, final concentration20 M). The reactions were mixed by aspirating several times and the samples then incubated in the dark (room temperature, 1 h). After this time 17 L of 4LDS sample buffer supplemented (6 mM BCN and 5% BME) was added and mixed by vortexing gently. Samples were incubated for 10 min before boiling at 90 C. for 10 min. Samples were analysed by 4-12% SDS-PAGE and fluorescent images were acquired using Typhoon Trio phosphoimager (GE Life Sciences).
(39) The same protocol for fluorescent labelling of the E. Coli proteins was applied for all tetrazine-dye conjugates.
Site-Specific Incorporation of 3 in HEK293 Cells and Chemoselective Labelling with Tetrazine Probes
Site Specific Incorporation of 3 in HEK Cells
(40) HEK293 Cells (ATCC CRL-1573) were plated on 24 well plates and grown to near confluence. The cells were transfected using Lipofectamine 2000 (Invitrogen) with the pMmPylS-mCherry-TAG-EGFP-HA construct and the p4CMVE-U6-PylT construct..sup.18 After 16 hrs growth with or without 1 mM 3 or with 1 mM 1 the cells were lysed on ice using RIPA buffer (Sigma). The lysates were spun down and the supernatant was added to 4LDS sample buffer (Life technologies). The samples were run out by SDS-PAGE, transferred to a nitrocellulose membrane and blotted using primary rat anti-HA(clone 3F10, Roche, No. 867 423) and mouse anti-FLAG (clone G191, Abnova, cat. MAB8183), the secondary antibodies were anti-rat (Invitrogen, A11077) and anti-mouse (Cell Signaling Technologies, No. 7076S).
Labelling Site-Specifically Incorporated 3 from HEK 293 Cells
(41) Adherent HEK293T cells (ATCC CRL-11268; 410.sup.6 per immunoprecipitation) were transfected with 7.5 g p4CMVE-U6-PylT and 7.5 g pPylRS-mCherry-TAG-EFGP-HA.sup.18 using TranslT-293 transfection reagent according to the manufacturer's protocol and cultured for 48 hours in DMEM/10% FBS, supplemented with 0.5 mM 1 or 2 mM 3 where indicated. Cells were washed twice with PBS and lysed on ice for 30 minutes in 1 mL Lysis Buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl (pH 8.0). After clarifying the lysate by centrifugation (10 min at 16000 g), HA-tagged proteins were captured using 50 L MACS HA-tag MicroBeads (Miltenyl Biotec) per transfection, washed with 0.5 mL RIPA (150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris HCl (pH 8.0) and 0.5 mL PBS (pH 7.4). The suspension of MicroBeads was incubated with 50 L PBS (pH 7.4), 20 M 4a for 1 hour and subsequently washed with 0.5 mL RIPA to remove excess dye. HA-tagged proteins were eluted from beads using SDS sample buffer and separated on a 4-12% Bis-Tris PAGE gel (Invitrogen), imaged using a Typhoon imager (GE Healthcare) and subsequently stained with DirectBlue or transferred for western blotting with Anti-HA-tag pAb-HRP-DirecT (MBL).
Expression and Purification of SfGFP from Mammalian Cells
(42) HEK293T were transfected in a 10 cm tissue culture dish with 15 ug DNA using PEI and incubated for 72 hours with 3 (0.5 M). Cells were washed twice with PBS and lysed in 1 mL RIPA buffer. Cleared lysate was added to 50 L GFP-Trap M (ChromoTek) and incubated for 4 h. Beads were washed with 1 mL RIPA, 1 mL PBS, 1 mL PBS+500 mM NaCl, 1 mL ddH2O and eluted in 1% Acetic Acid/ddH2O. Purified protein was labeled with 2 M 4a for 4 h and loaded on a 4-12% Bis-Tris PAGE gel. Fluorescence of 4a-labeled sfGFP was detected on a Typhoon imager and gel was stained subsequently with DirectBlue.
Fly Plasmids, Transgenicflies and Culture
(43) For all fly experiments no randomisation or blinding was used within this study
Plasmid Construction for Transgenic Fly Line Generation
(44) The PyltRNA.sub.CUA anticodon was mutated using the QuikChange mutagenesis kit and pSG108 (pJet 1.2-U6-PylT, gift from S. Greiss) as a template. This contains the PylT gene without its 3 terminal CCA fused to the Drosophila U6-b promoter. Primers FMT19 and FMT20 were used to generate PyltRNA.sub.TGC to decode alanine codons (creating pFT18); primers FMT23 and FMT24 were used to generate PyltRNAccr to decode serine codons (creating pFT20); primers FMT27 and FMT28 were used to generate PyltRNA.sub.CAG to decode leucine codons (creating pFT22) and primers FMT29 and FMT30 were used to generate PyltRNA.sub.CAT to decode methionine codons (creating pFT23). The mutated tRNA expression cassettes were subcloned from pFT18, pFT20, pFT22 and pFT23 into pUC18 using EcoRI and HinDIII then multimerised using AsiSI, BamHI and BglII to create 2, then 4 copies of the tRNA. The 4 copy versions of the tRNA cassette were subcloned into pSG118 using AsiSI and MluI to create pFT58 (Ala), pFT60 (Ser), pFT62 (Leu) and pFT63 (Met). pSG118 contains the M. mazei PylRS gene..sup.20
Fly Lines and Culture Conditions
(45) Transgenic lines were created by P element insertion using a Drosophila embryo injection service (BestGene Inc.). Lines were generated using the following plasmids: pFT58 (Ala), pFT60 (Ser), pFT62 (Leu) and pFT63 (Met). nos-Gal4-VP16 (Bloomington 4937) and MS1096-Gal4 (Bloomington 8860) were used as Gal4 drivers. All flies were grown at 25 C. on standard Iberian medium. Flies were fed unnatural amino acids by mixing dried yeast with the appropriate concentration of amino acid (usually 10 mM) diluted in dH.sub.2O to make a paste. Ovaries were prepared from females that were grown on Iberian fly food supplemented with a yeast paste with or without the amino acid for a minimum of 48 hours. For proteome labelling experiments transgenic male flies of constructs FT58, FT60, FT62 and FT63 were crossed with nos-vp16-GAL4 virgins to generate FT58/nos-vp16-GAL4, FT60/nos-vp16-GAL4, FT62/nos-vp16-GAL4 and FT63/nos-vp16-GAL4 respectively.
Site Specific Incorporation of 3 in D. melanogaster
Luciferase Assays
(46) Ovaries from 10 females of Triple Rep-L flies recombined with nos-Gal4-VP16 fed 3, 1 or no amino acid were dissected in 100 l 1 Passive lysis buffer and processed for luciferase assays as previously described.sup.20.
Immunoprecipitation and Labelling of Site Specifically Incorporated 3
(47) Ovaries from 100 (for control and 3) or 500 (for 1) females were dissected in PBS then lysed in 300 or 1500 l RIPA buffer containing ix complete protease inhibitor cocktail (Roche). A sample was taken into 4LDS buffer as a total lysate control then the remainder was used for immunoprecipitation with GFP-TRAP agarose beads (Chromotek) following the manufacturer's instructions. The total volume of the IP was 3 ml. After overnight incubation, the beads were washed 2 with RIPA buffer then 2 with PBS. For tetrazine labeling, the beads were resuspended in 200 l PBS+4 M 4g and incubated for 2 hours on a roller at RT. The beads were washed 3 times with 500 L of wash buffer then resuspended in 4LDS sample buffer.
Example 1Synthesis of N.SUP..-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-L-lysine 3
(48) A class of reaction useful in protein labelling is the very rapid and specific inverse electron demand Diels-Alder reaction between strained alkenes (or alkynes) and tetrazines..sup.21-25
(49) While we, and others, have previously encoded unnatural amino acids bearing strained alkenes, alkynes and tetrazines via genetic code expansion and demonstrated their use for site-specific protein labelling via inverse electron demand Diels-Alder reactions,.sup.26-30 all the molecules used to date are rather large. We have previously shown that a variety of carbamate derivatives of lysine are good substrates for PylRS,.sup.31 and it has been demonstrated that 1,3 disubstituted cyclopropenes, unlike 3,3 disubstituted cyclopropenes,.sup.32,24 react efficiently with tetrazines..sup.22 We therefore designed and synthesized a carbamate derivative of lysine, bearing a 1,3 disubstituted cyclopropene (N.sup.-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-L-lysine 3,
Synthesis of Methylcycloprop-2-en-1-yl}methoxy)carbonyl]-L-lysine (3)
(50) ##STR00004##
i. Ethyl 2-methylcycloprop-2-ene-1-carboxylate S1
(51) A 100 mL 2-neck round bottom flask was charged with CH.sub.2Cl.sub.2 (2 mL) and rhodium acetate (442 mg, 1 mmol, 0.05 eq), and fitted with a dry ice condenser. Propyne (approx. 10 mL) was condensed into the rhodium acetate suspension and the flask lowered into a water bath (20 C.), a steady reflux of propyne was obtained. Ethyl diazoacetate (2.1 mL, 20 mmol, 1 eq) was added to the stirred propyne solution drop-wise over 1 h using a syringe pump. The reaction was stirred at room temperature for a further 10 minutes whereby TLC analysis showed the reaction to be complete by after this time. The cyclopropene product was then purified by silica gel flash column chromatography eluting with pentane and diethyl ether (90:10). This gave the desired product S1 as a colourless volatile liquid (1.9 g, 75% yield). .sup.1H NMR analysis .sub.H (400 MHz, CDCl.sub.3) 6.35 (1H, t, J 1.4), 4.18-4.09 (2H, m), 2.16 (3H, d, J 1.3), 2.12 (1H, d, J 1.6), 1.26 (3H, t, J 7.1); LRMS m/z (ES.sup.+) 127.2 [M+H].sup.+.
(52) These values are in good agreement with literature. {Liao, 2004 #1}
ii. and iii. (2-Methylcycloprop-2-en-1-yl)methyl (4-nitrophenyl) Carbonate S3
(53) DIBAL-H (22.5 mL of a 1M solution in CH.sub.2Cl.sub.2, 22.5 mmol, 1.5 eq) was added drop-wise to a stirred solution of cyclopropene ester S1 (1.9 g, 15 mmol, 1 eq) in CH.sub.2Cl.sub.2 (15 mL) at 10 C. The reaction was stirred at 10 C. for 20 minutes before quenching with the cautious addition of H.sub.2O (1 mL), then NaOH (1 mL of a 1 M solution in H.sub.2O) and H.sub.2O (2.3 mL). The mixture was stirred for a further 2 h at room temperature before it was dried (Na.sub.2SO.sub.4) and filtered. Hunig's base (3.9 mL, 22.5 mmol, 1.5 eq) was added to the filtrate (containing crude cyclopropene alcohol S2) followed by the addition of 4-nitrophenyl chloroformate (3.3 g, 16.5 mmol, 1.1 eq). After stirring at room temperature for 18 hours a significant colourless precipitate formed, and TLC analysis showed complete consumption of the crude cyclopropene alcohol S2. The reaction was diluted with CH.sub.2Cl.sub.2 and then dry loaded onto silica gel, whereby the activated carbonate S3 was purified by silica gel column chromatography eluting with ethyl acetate and hexane (20:80). This gave the desired cyclopropene carbonate S3 as a colourless oil (2.7 g, 73% yield over 2 steps). .sup.1H NMR analysis .sub.H (400 MHz, CDCl.sub.3) 8.28 (2H, d, J 9.2), 7.39 (2H, d, J 9.2), 6.62 (1H, s), 4.21 (1H, dd, J 10.9, 5.3), 4.14 (1H, dd, J 10.9, 5.3), 2.18 (3H, d, 1.3), 1.78 (1H, td, J 5.3, 1.3).
iv. N.SUP..-(Fmoc)-N.SUP..-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-L-lysine S4
(54) Fmoc-Lys-OH.HCl (6.7 g, 16.5 mmol, 1.5 eq) was dissolved in THF (30 mL) and DMF (10 mL), to this solution was added Hnig's base (9.0 mL, 55.0 mmol, 5 eq) followed by cyclopropene carbonate S3 (2.7 g, 11.0 mmol, 1 eq) an immediate yellow coloration was observed upon addition of the carbonate. The reaction was stirred at room temperature for 6 hours and was adjudged complete by the consumption of starting material after this time as shown by TLC analysis. The crude reaction mixture was dry loaded onto silica gel and the major product purified by silica gel column chromatography eluting with ethyl acetate, hexane and acetic acid (50:49:1 then 99:0:1). This gave the desired product S4 as a colourless gum (4.3 g, 82% yield). .sup.1H NMR analysis .sub.H (400 MHz, CDCl.sub.3) 7.77 (2H, t, J 7.6), 7.65-7.55 (2H, m), 7.39 (2H, t, J 7.6), 7.31 (2H, t, J 7.3), 6.54 (1H, s), 5.68-5.57 (1H, m), 4.84 (1H, br-s), 4.44-4.32 (2H, m), 4.22 (1H, t, J 7.0), 3.98-3.87 (1H, m), 3.17-3.09 (2H, m), 2.15-2.06 (6H, m), 1.99-1.86 (1H, m), 1.84-1.70 (1H, m), 1.68-1.59 (1H, m), 1.58-1.34 (2H, m); LRMS m/z (ES.sup.+) 479.3 [M+H].sup.+, 501.3 [M+Na].sup.+, m/z (ES.sup.) 477.2 [MH].sup..
N.SUP..-[({2-methylcycloprop-2-en-1-yl}methoxy)carbonyl]-L-lysine 3
(55) N.sup.-(Fmoc)-N.sup.-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-L-lysine S4 (3.5 g, 7.0 mmol, 1 eq) was dissolved in THF and H.sub.2O (3:1 40 mL), to this solution was added sodium hydroxide (0.9 g, 22.6 mmol, 3.1 eq). The reaction was stirred at room temperature for 8 hours after which time the reaction was adjudged complete by LC-MS analysis. The reaction mixture was diluted with H.sub.2O (100 mL) and the pH adjusted to 5 by the addition of HCl (1M). The aqueous solution was washed with Et.sub.2O (5100 mL), then concentrated to dryness yielding a colourless solid. The solid was purified by preparative HPLC, the product fractions were combined and the solvent removed by freeze-drying. This gave N.sup.-[({2-methylcycloprop-2-en-1-yl}methoxy)carbonyl]-L-lysine 3 as a colourless solid. .sub.H (400 MHz, D.sub.2O) 6.45 (1H, s), 3.90-3.61 (2H, m), 3.09 (1H, t, J 6.4), 2.98-2.86 (2H, m), 1.92 (3H, s), 1.52-1.37 (2H, m), 1.37-1.22 (2H, m), 1.21-1.08 (2H, m), 0.83 (1H, d, J 5.2). LRMS m/z (ES.sup.+) 257.2 [M+H].sup.+, m/z (ES.sup.) 255.2 [MH].sup.. .sub.C (100 MHz, D.sub.2O) 101.1 (CH), 72.3 (CH.sub.2), 55.9 (CH), 40.2 (CH.sub.2), 34.3 (CH.sub.2), 28.9 (CH.sub.2), 20.3 (CH.sub.2), 16.6 (CH.sub.3), 10.8 (CH) HRMS (ES.sup.+) Found: (M+Na).sup.+ 279.1302. C.sub.12H.sub.20O.sub.4N.sub.2Na required M.sup.+, 279.1315.
Example 2Encoding the Site-Specific Incorporation of 3 in E. coli
(56) We demonstrated that 3 is efficiently and site-specifically incorporated into recombinant proteins in response to the amber codon using the PylRSARNA.sub.CUA pair and an SfGFP gene bearing an amber codon at position 150 (Supplementary
(57) Since PylRS does not recognize the anticodon of its cognate tRNA.sup.34 it is possible to alter the anticodon of this tRNA to decode distinct codons. We created a new tRNA in which the anticodon of PyltRNA.sub.CUA was converted from CUA to UUU (Supplementary Table 1), to decode a set of lysine codons. We added 0.1 mM 3 to cells containing PylRS, PyltRNA.sub.UUU, and the gene for T4 lysozyme. Following expression of T4 lysozyme we detected proteins in the lysate bearing 3 with the tetrazine probe 4a (20 microM 1 h, Supplementary
Example 3Genetic Encoding of 3 in Human Cells
(58) Full-length mCherry-3-GFP-HA was expressed in HEK293 cells carrying the PylRSARNA.sub.CUA pair and mCherry-TAG-EGFP-HA (a fusion between the mCherry gene and the EGFP gene with a C-terminal HA tag, separated by the amber stop codon (TAG))..sup.18 Full-length protein was detected only in the presence of the 3 (
Example 4Genetic Encoding of 3 in D. melanogaster
(59) We demonstrated that 3 can be site specifically incorporated into proteins in D. melanogaster. To achieve this, we used flies containing the PylRS/tRNA.sub.CUA pair (with the tRNA expressed ubiquitously from a U6 promoter and UAS-PylRS expression directed to ovaries using a nos-vp16-GAL4 driver), and a dual luciferase reporter bearing an amber codon between firefly and renilla luciferase..sup.20 We observe a strong luciferase signal that is dependent on the addition of 1 or 3, and the dual luciferase signal is larger with 3. These experiments demonstrate that 3 is taken up by flies and is more efficiently incorporated in vivo in response to an amber codon than 1 (
Example 5Synthesis of Tetrazine-BODIPY FL 4d
(60) ##STR00005##
(61) Boc-protected Tetrazine S6 was synthesized using the procedure reported earlier.sup.6. 4M HCl in dioxane (500 L, 2.0 mmol) was added to a stirring solution of Tetrazine S5 (8 mg, 0.02 mmol) in DCM (500 L). The reaction was carried out for 2 h at room temperature and subsequently the solvent was removed under reduced pressure to yield primary amine hydrochloride S6 as a pink solid (6 mg, 0.02 mmol, 100%). The compound was directly used in the next step without any further purification.
ii. 4d
(62) BODIPY FL succinimidyl ester (5 mg, 0.013 mmol, Life technologies) and Hnig's base (50 l, 2.8 mmol) were added to the solution of Tetrazine-amine S2 (6 mg, 0.02 mmol) in dry DMF (1 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with 4 ml of water and the product was purified by semi-preparative reverse phase HPLC using a gradient from 10% to 90% of buffer B in buffer A (buffer A: H.sub.2O; bufferB: acetonitrile). The identity and purity of the tetrazine-BODIPY FL conjugate 4d was confirmed by LC-MS. ESI-MS: [MH].sup., calcd. 581.38, found 581.2.
Summary of Examples 1 to 5
(63) We have characterized the synthesis of, and the genetically encoded, site-specific incorporation of a cyclopropene containing amino acid 3, and demonstrated the quantitative labelling of 3, with tetrazine probes, in proteins expressed in E. coli, mammalian cells and D. melanogaster, thereby showing the widespread utility and industrial application of the present invention.
SUPPLEMENTARY REFERENCES TO EXAMPLES 1 to 5
(64) 1. Gautier, A. et al. Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells. Journal of the American Chemical Society 132, 4086-4088 (2010). 2. Karp, N A, Kreil, D. P. & Lilley, K. S. Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4, 1421-1432 (2004). 3. Karp, N. A. & Lilley, K. S. Design and analysis issues in quantitative proteomics studies. Proteomics 7 Suppl 1, 42-50 (2007). 4. Lilley, K. S. in Current Protocols in Protein Science (John Wiley & Sons, Inc., 2001). 5. Von Stetina, J. R., Lafever, K. S., Rubin, M. & Drummond-Barbosa, D. A Genetic Screen for Dominant Enhancers of the Cell-Cycle Regulator alpha-Endosulfine Identifies Matrimony as a Strong Functional Interactor in Drosophila. G3 (Bethesda) 1, 607-613 (2011). 6. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 4, 298-304 (2012).
Example 6Dual Labelling of Proteins
(65) The ability to attach two distinct molecules to programmed sites in proteins will facilitate a variety of applications including FRET.sup.1,2 to study protein structure, conformation and dynamics. Several approaches for doubly labeling proteins have been reported. One approach relies on the installation of one unnatural amino acid that is specifically labeled in combination with cysteine thiol labeling, but this approach is generally limited to proteins that do not contain free thiols..sup.3,4 Chemical ligation approaches can be combined with the genetic encoding of a single unnatural amino acid for protein labeling,.sup.5 but this may limit the size and/or sites that may be labeled. Perhaps the most generally applicable approach for protein double labelling is based on the genetic incorporation of two distinct amino acids in response to two distinct codons introduced at user defined sites in the gene of interest.
(66) An ideal strategy for dual labeling requires i) the efficient, cellular, incorporation of two distinct unnatural amino acids into a protein that can be labelled in mutually orthogonal reactions, and the development of mutually orthogonal reactions that allow the simultaneous addition of two molecules to the protein for rapid, quantitative labelling of the protein in aqueous media at physiological pH, temperature and pressure.
(67) Scheme A (
(68) The cellular, genetically directed incorporation of two distinct unnatural amino acids into proteins has been demonstrated in response to an amber and quadruplet codon,.sup.6 two distinct stop codons,.sup.7,8 or two distinct quadruplet codons..sup.9 We previously demonstrated the evolution of an orthogonal ribosome (ribo-Q1) that efficiently reads quadruplet codons and amber codons on orthogonal mRNA using cognate extended anticodon tRNAs or amber suppressors respectively..sup.6 We demonstrated that the pyrrolysyl-tRNA synthetase/tRNA pair and synthetically evolved derivatives of the MjTyrRS/tRNA pair are mutually orthogonal in their aminoacylation specificity and can be used to direct the incorporation of pairs of unnatural amino acids in response to amber and quadruplet codons..sup.6 We recently described several major advances in this system, including the evolution of a series of quadruplet decoding tRNAs based on the pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair that efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons using the evolved orthogonal translation machinery..sup.9 We demonstrated the very efficient incorporation of a matrix of pairs of unnatural amino acids using the evolved PylRS/tRNA.sub.UACU pair and derivatives of the MjTyrRS/tRNA.sub.CUA pair with orthogonal messages bearing TAG and AGTA codons and ribo-Q1..sup.9
(69) A limited range of chemistries have been investigated for the double labeling of proteins containing pairs of unnatural amino acids. The incorporation of azide- and alkyne-containing amino acids, and their non-quantitative labeling with alkyne and azide based fluorophores has been reported.sup.7, but this is not ideal for double labeling of proteins; if the encoded azide and alkyne are in proximity they can react to form a triazole in the protein, a strategy which allows genetically directed protein stapling,.sup.6 but precludes labeling with probes. Moreover, an efficient one-pot reaction is not feasible because of the reaction between azide- and alkyne-bearing probes with each other. The incorporation of ketone and azide containing amino acids has been reported,.sup.8,10 which allows one-pot reaction of the encoded ketone with alpha effect nucleophiles, and the azides with alkyne probes..sup.10 However this approach is problematic because encoded azides are subject to reduction in many proteins when expressed in E. coli,.sup.8,11 which will prevents quantitative labeling. Moreover, ketone labeling with alpha effect nucleophiles is very slow (rate constant approximately 10.sup.4 M.sup.1 s.sup.1) and the reaction is optimal at pH4-5.5,.sup.12 which limits its utility for many proteins that are denatured or precipitate when kept for long periods under acidic conditions. We recently genetically installed a deactivated tetrazine containing amino acid.sup.13 and a norbornene containing amino acid.sup.14-16 into proteins using our optimized orthogonal translation system..sup.9 Because the rate of inverse electron demand Diels Alder reaction between the deactivated tetrazine and norbornene is very slow, but the tetrazine can react with bicyclononyne based probes and the norbornene can react with activated tetrazine probes we were able to use this approach to specifically and quantitatively double label proteins..sup.9 While this approach has the advantage of proceeding in aqueous media at physiological pH, temperature and pressure; it does require sequential labeling steps (to avoid inverse electron Demand reactions between probes), each of which takes several hours, with purification between steps. All approaches reported to date for doubly labeling proteins at genetically encoded unnatural amino acids take tens of hours to days to reach completion.
(70) An ideal approach to double label proteins would allow rapid one-pot labeling of genetically installed bio-orthogonal functional groups, proceed rapidly in aqueous media at physiological pH, temperature and pressure and be implemented simply by adding the labeling reagents to a recombinant protein bearing the site specifically incorporated bioorthogonal groups. A promising pair of mutually orthogonal reactions for one-pot labeling under aqueous conditions at physiological pH are the Cu(I)-catalysed 3+2 cycloaddition between azides and terminal alkynes,.sup.17 and the inverse electron demand Diels Alder reaction of a strained alkenes and a tetrazine.sup.18-23(
(71) We demonstrated in examples 1 to 5 that a 1,3 disubstituted cyclopropene containing amino acid, 2 (referred to as 3 in examples 1 to 5 and elsewhere in this document), can be efficiently and site specifically incorporated into proteins using the PylRS/tRNA.sub.CUA pair..sup.25 This amino acid, unlike the 3,3 disubstituted cyclopropene incorporated for photoclickreactions,.sup.26 reacts with tetrazines.sup.19,27 with on-protein rate constants of 27 M.sup.1 s.sup.1..sup.25 Here we demonstrate the efficient genetic encoding of a terminal alkyne containing amino acid 1 and a cyclopropene containing amino acid 2 into a single protein and their rapid, quantitative, one-pot labeling with azide and tetrazine probes (
(72) Proteins containing either 1 or 2 were overexpressed to examine the specificity of the orthogonality of the proposed labeling reactions. A fusion protein of glutathione-S-transferase and calmodulin (GST-CaM) with amino acid 1 at position 1 in calmodulin was expressed from cells containing ribo-Q1 (an evolved orthogonal ribosome.sup.6,28,29), O-gst-cam.sub.1TAG (a fusion gene between glutathione-S-transferase (gst) and calmodulin (cam) on an orthogonal message.sup.30 in which the first codon of cam is replaced with a TAG codon), and MjPrpRS/tRNA.sub.CUA (a synthetase/tRNA pair developed for incorporating t in response to the TAG codon).sup.31 grown in the presence of (4 mM). The GST tag was subsequently removed by cleavage using thrombin at an engineered thrombin-cleavage site between GST and CaM. CaM1.sub.1 (CaM containing 1 at position 1, 100 pmole) was labelled with the azide containing fluorophore 3 (2 nmole), in a Cu (I)-catalysed click reaction. The reaction was quantitative as judged by both the quantitative shift of the fluorescently labelled protein by SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) (
(73) The cyclopropene containing amino acid, 2, was site specifically incorporated at position 40 of calmodulin. The modified protein was expressed in cells bearing the PylRS/tRNA.sub.CUA (that efficiently directs the site specific incorporation of 2),.sup.25 ribo-Q1, and O-gst-cam.sub.40TAG grown in the presence of 2 (1 mM). CaM2.sub.40 (100 pmol) (obtained after thrombin cleavage of the GST tag) was labelled with the tetrazine containing fluorophore 4 (2 nmole). The reaction was quantitative as judged by both the quantitative shift of the fluorescently labelled protein by SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) (
(74) Next we investigated labeling 1 and 2 within the same protein. We site-specifically incorporated 1 and 2 at positions 1 and 40 of calmodulin to produce CaM1.sub.12.sub.40 (
(75) To determine the time required to quantitatively label CaM1.sub.12.sub.40 with azide 3 or tetrazine 4 we incubated 100 pmol of CaM1.sub.12.sub.40 with 2 nmol of either 3 or 4 and followed each reaction by both mobility shift on SDS-PAGE and fluorescent imaging upon labeling (
(76) Next we investigated the labeling of CaM1.sub.12.sub.40 with both 3 and 4 (
(77) In summary, in this example we show an efficient and rapid protocol for expressing recombinant proteins bearing a site specifically incorporated alkyne and a site specifically incorporated cyclopropene. We demonstrate that the inverse electron demand Diels Alder reaction of an encoded 1,3 disubstituted cyclopropene and tetrazine probe, and the 3+2 cycloaddition reaction of the encoded alkyne and azide probe are mutually orthogonal to each other and to the functional groups in proteins. By combining the genetic encoding of an alkyne and a cyclopropene in a single protein and labelling with the mutually orthogonal reactions we demonstrate the concerted, one-pot rapid double labeling of a protein in aqueous media at physiological pH and temperature. This strategy has utility for doubly labeling proteins for a variety of studies and applications, and may be extended to the double labeling of diverse molecules in diverse cells and organisms.
(78) Note on example 6: The chemical designations in example 6 and in the corresponding figures (drawings) discussed in example 6 are self-contained and apply only to example 6. Discussion of chemical designations in the rest of this document are consistent with the exception of example 6. For example, the skilled reader will immediately appreciate that compound 2 of example 6 corresponds to compound 3 in the rest of this document (i.e. the exemplary cyclopropene amino acid of the invention). Compounds 3 and 4 of example 6 are tetrazine compounds.
REFERENCES TO EXAMPLE 6
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