BIOCONJUGATION TO QUANTUM DOTS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/312,785, filed on March 11, 2010, the entire contents of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.

5RO1CA126642-02, 5-U54-CA119349-05, U01-HL080731, and T32-CA79443 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

The present disclosure provides compositions and methods using bioorthogonal inverse electron demand Diels-Alder cycloaddition reactions for rapid and specific coupling of organic compounds to quantum dots (QDs).

BACKGROUND

Conventional QD conjugation methods typically rely on functional groups such as amines, carboxylic acids, and thiols that are known to interact with the QD surface.

Surface coordination of functional groups can limit the number of available groups for further coupling, resulting in low conjugation efficiencies. An attractive alternative is to employ coupling chemistry that requires functional groups that do not coordinate to the QD surface. Click chemistries, such as the popular copper catalyzed azide-alkyne cycloaddition, are potential alternative conjugation strategies. However, for catalyzed "click" chemistry, the requisite copper catalyst irreversibly quenches QD fluorescence. Additionally, catalyst- free strain-promoted click reactions are limited by solubility of substrates in buffer and serum and tedious syntheses. SUMMARY

The present disclosure provides methods and compositions based on a

bioorthogonal inverse electron demand Diels- Alder cycloaddition reaction for rapid and specific coupling of organic compounds to QDs. This Diels-Alder reaction connects the two components of the reaction, a diene and a dienophile and thus conjugates the organic compound with the QD. The diene, e.g. a functionalized tetrazine, is attached to the organic compound, and the dienophile e.g. a functionalized norbornene, is attached to the QDs. The QD is coupled with a copolymer to provide water solubility, functionality, and QD binding. The bioorthogonal chemistry platform can be used extracellularly or intracellular ly, in vivo or in vitro.

In one aspect, the present disclosure provides compositions comprising: a copolymer comprising poly (ethylene) glycols, amino-poly(ethylene)glycoln, and imidazole linked to an alkene.

In another aspect, the present disclosure provides compositions comprising: a quantum dot coupled with a copolymer comprising poly(ethylene) glycols, amino- poly(ethylene)glycoln, and imidazole, and wherein the copolymer is linked to an alkene.

In some embodiments, the compositions described herein further comprise an organic compound linked to the alkene by an inverse electron demand Diels-Alder cycloaddition reaction.

In some embodiments, the inverse electron demand Diels-Alder cycloaddition reaction occurs ex vivo or in vivo. In some embodiments, epidermal growth factors (EGFRs) are labeled with an organic compound, e.g., epidermal growth factor (EGF), either directly through the use of pre-formed QD-EGF conjugates or by performing in situ conjugation of the norbornene-coupled QDs to tetrazine modified EGFs on the cell surface. For example, the QD-EGF conjugates can be formed by the inverse electron demand Diels-Alder cycloaddition reaction first and then directly labeling the cell, or the inverse electron demand Diels-Alder cycloaddition reaction can occur in situ on the cell surface. In some embodiments, the organic compound is an fluorescent materials, luminescent materials, bioluminescent materials, growth factors, antibodies, peptides, proteins, DNA, or RNA.

In some embodiments, the copolymer comprises a first reactive group and the alkene comprises a second reactive group; wherein the first and second reactive groups are each selected from an amine, a carboxylic acid, an activated ester, alkyne, or a triazole; and reaction of the first and second reactive groups couples the copolymer to the norbornene compound.

In some embodiments, the first and second components are an amine and an activated ester and react to link the copolymer with the norbornene to form an amide bond.

In some embodiments, the copolymer comprises poly(ethylene) glycols, amino- poly(ethylene)glycoln, and imidazole.

In some embodiments, the copolymer comprises 30% poly(ethylene) glycol (PEG12), 20% amino-PEGn, and 50%> imidazole groups.

In some embodiments, the alkene can be a strained alkene, e.g. , a norbornene compound or a trans-cyclooctene.

In some embodiments, the copolymer comprises poly(ethylene) glycol^, amino- poly(ethylene)glycoln, and imidazole and has an amino group as the first reactive group; and

the alkene is having an activated NHS ester as the second reactive group. The activated NHS-ester on the alkene reacts with the amino group on the copolymer to form an amide bond linking the alkene to the copolymer.

In another aspect, the present disclosure provides methods of linking a copolymer comprising poly(ethylene) glycols, amino-poly(efhylene)glycoln, and imidazole to an alkene, the method comprising: contacting the copolymer having a first reactive group with the alkene having a second reactive group, wherein: the first and second reactive groups are selected from the group consisting of: an amine, a carboxylic acid, and an activated NHS-ester.

In some embodiments, the copolymer is coupled to a quantum dot before or after the copolymer is linked to the alkene.

In some embodiments, the copolymer is coupled to the quantum dot after the copolymer is linked to the alkene.

In some embodiments, the alkene is further reacted with a diene attached to an organic compound.

In some embodiments, the alkene is a norbornene compound and the diene is a tetrazine, and the norbornene compound and the tetrazine are participants in the inverse electron demand Diels- Alder reaction.

In some embodiments, the organic compound is selected from the group consisting of: fluorescent materials, luminescent materials, bioluminescent materials, growth factors, antibodies, peptides, proteins, DNA, and RNA.

DESCRIPTION OF DRAWINGS

FIG. lA is a scheme depicting the conjugation of norbornene to 20% NH ₂ -PIL polymer.

FIG IB is a scheme depicting the conjugation of Alexa with Quantum Dots570 using [3-(4-benzylamino)-l,2,4,5-tetrazine] (BAT)+norbornene chemistry.

FIG. 1C is a graph depicting the absorbance spectra of QD-Alexa conjugates which were prepared by mixing carrying concentrations of the dye and purifying by gel filtration chromatography and multiple dialysis.

FIG. ID is a graph depicting calculated Alexa to QD ratios for the purified conjugates.

FIG. 2 is a graph depicting the probing of free amines in different polymer samples using fluorescamine. Square points depict poly(amino-PEGn) ₂ o _% -PIL, circle points depict poly(PEGi ₂ )-PIL, triangle points are after converting the amine of poly(amino-PEGn)2o _% -PIL to norbornene (NB-PIL). Fluorescence of NB-PIL being similar level as poly(PEGi ₂ )-PIL proves the conversion was complete.

FIG. 3 is a transmission electron microscopy (TEM) image of CdSe(CdS) with inorganic size ~4.6 nm.

FIG. 4A is a scheme depicting conjugation of NHS-activated BAT to EGF.

FIG. 4B is a scheme depicting the labeling of cells with pre-conjugated QDs to antibodies.

FIG. 4C is a scheme depicting the in situ conjugation of QDs to antibodies on cells.

FIGs. 5A-D are fluorescence images depicting the labeling of antibody-QD conjugates to A431 (skin cancer) cells. FIGs. 5A-D top images: QD fluorescence at 605 nm with excitation at 488 nm. FIGs. 5A-D bottom images: corresponding DIC images (scale bar 10 μιη). Cells were targeted either (5B) with the preformed 50 nM QD-EGF complex for single QD tracking or (5D) by performing in situ conjugation of 800 nM QDs to EGFs for efficient cell labeling. (5A) and (5C) are control experiment with

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for the bioorthogonal and modular conjugation of an organic compound to quantum dots (QDs). These methods include the use of bioconjugation through bioorthogonal chemistry, e.g. the inverse electron demand Diels-Alder reaction, to couple an organic compound to QDs. For example, the organic compound can be functionalized with a diene, and the dienophile can be attached to the QDs through a copolymer which is coupled to the QD. Alternatively, the dienophile can be attached to the organic compound and the diene can be attached to the QDs through the copolymer which is coupled to the QD. The QDs are coupled with the copolymer, e.g., a polymeric imidazole ligand (PIL), to provide for water solubilization, functionalization, e.g., a reactive group such as an amino group, and QD binding. The methods and compositions can be used, e.g., in vivo and in vitro, both extracellularly or intracellularly, as well as in assays such as cell free assays. By virtue of their design the composition and methods described herein possess a number of advantages. First, efficient conjugation methods utilizing the inverse electron demand Diels Alder cycloaddition reaction between functionalized tetrazines and norbomenes provide for rapid kinetics. The rapid kinetics combined with tolerance of the reaction to functional groups abundant in cells, e.g. amines, allows the labeling of proteins of interest on cells in situ. Additionally, the conjugation approach presented herein is modular and can be extended to many biological imaging applications, as tetrazine and norbomene functionalities can be easily conjugated to carboxylic acid or amine containing molecules.

Diels- Alder Pairs

The compositions and methods described herein include the use of Diels- Alder pairs that include a diene and a dienophile. The inverse electron demand Diels- Alder cycloaddition reaction of a diene (e.g., a substituted tetrazine) with a dienophile (e.g., an alkene or alkyne), produces an unstable cycloadduct which subsequently undergoes a retro-Diels-Alder cycloaddition reaction to produce dinitrogen (N ₂ ) as a byproduct and the desired dihydropyrazine (after reaction with an alkene) or pyrazine (after reaction with an alkyne) products (Scheme 1). The dihydropyrazine product may undergo an additional oxidation step to generate the corresponding pyrazine.

Bioorthogonal Chemistry

Bioconjugation methods using inverse electron demand Diels-Alder

cycloadditions between tetrazines and highly strained dienophiles such as norbomene and trans-cyclooctene are known in the literature, however, the tetrazine used has limited stability in aqueous media. (Blackman et al, 2008, J. Am. Chem. Soc, 130, 13518-9; Devaraj et al, 2009, Angew Chem Int Ed Engl, 48, 7013-6; Devaraj et al, 2008, Bioconjug Chem, 19, 2297-9; Pipkom et al, 2009, J Pept Sci, 15, 235-41). A tetrazine derivative, such as [3-(4-benzylamino)-l,2,4,5-tetrazine] (BAT), can be used that shows good stability in buffer and serum and a high reaction rate when reacted with norbomene (Scheme 1, 2 M ^' V ¹ at 20 °C) or trarcs-cyclooctene (-6000 M ^' V ¹ at 37 °C). See also e.g., WO 2010/051530 which is incorporated by reference in its entirety, and describes both dienes and dienophiles for the inverse electron demand Diels- Alder cycloaddition reaction. This extremely fast rate constant allows for the labeling of extracellular targets at low nanomolar concentrations of tetrazine labeling agent, concentrations that are sufficiently low to allow for real-time imaging of probe accumulation.

Scheme 1

For example, the bio-orthogonal inverse electron demand Diels- Alder reaction can be tailored to provide a straightforward method for the rapid, specific covalent coupling of an organic compound to QDs for labeling and imaging of proteins of interest on cells.

In some embodiments, an organic compound is chemically attached to the diene, e.g., to the tetrazine. In some embodiments, the organic compound carries a functional group such as an amine, alcohol, carboxylic acid or ester, or other group of atoms on the organic compound that can undergo a chemical reaction allowing for attachment to the diene, e.g., to the tetrazine. Additionally, the dienophile (which can be or include, e.g., an alkene, alkyne, nitroso, carbonyl or imine) possesses a functional group for attachment to the copolymer which may or may not be coupled to the QDs. Thus, the reactive functional group on the copolymer or copolymer already coupled to the QDs and the dienophile undergo a chemical reaction to form a chemical bond between the two functional groups to form a dienophile/copolymer or dienophile/copolymer/QD conjugates.

In some embodiments, the copolymer (which may or may not be already coupled to the QDs) can be linked to the dienophile through an amide bond.

In some embodiments, the organic compound is attached to the diene through an amide bond. Dienes

Dienes useful in the present disclosure include, but are not limited to, aromatic ring systems that contain two adjacent nitrogen atoms, for example, tetrazines, pyridazines, substituted or unsubstituted 1,2-diazines. Other 1,2-diazines can include 1,2-diazines annelated to a second π-electron-deficient aromatic ring such as pyrido[3,4- d]pyridazines, pyridazino[4,5-d]pyridazines, and 1,2,4-triazines. Pyridazines can also be fused with a five-membered heterocycle such as imidazo[4,5-d]pyridazines and 1,2,3- triazolo[4,5-d]pyridazines. In some embodiments, the diene can be a substituted tetrazine or other heteroaromatic ring system with at least two nitrogens adjacent to each other and which is a highly reactive participant in the inverse electron demand Diels-Alder reaction.

In some embodiments, the diene is an asymmetrical tetrazine, e.g., 3-(p- Benzylamino)-l,2,4,5-tetrazine, which has been functionalized with an NHS-activated linker (1).

(1)

Dienophiles

Dienophiles useful in the present compositions and methods include, but are not limited to, carbon containing dienophiles such as alkenes or alkynes, or compounds containing nitroso, carbonyl, or imine groups. In some embodiments, the dienophile is a strained dienophile such as a norbornene.

As used herein, a "strained" dienophile has a dihedral angle that deviates from the idealized 180 degree dihedral angle. As used herein, "alkenes" refer to an alkyl group having one or more double carbon-carbon bonds such as an ethylene, propylene, and the like. Alkenes can also include cyclic, ring-strained alkenes such as trans-cyclooctene or norbomene carrying a double bond which induces significant ring strain and is thus highly reactive. Alkenes can also include more complex structures such as indoles and azaindoles, or electron rich enamines. Heterodienophiles containing carbonyl, nitroso or imine groups can also be used. In some embodiments, the dienophile is a norbomene. In some embodiments, the alkene is a norbomene functionalized with a carboxylic acid or NHS-activated ester for attachment to the QDs (2).

In some embodiments, the norbomene is functionalized with a carboxylic acid that can be NHS-activated and the QDs is coupled with a copolymer comprising an amine functional group and the two functional groups react to form an amide bond thereby linking the norbomene to the copolymer which is coupled to the QD. Alternatively, the amine can be located on the norbomene and the carboxylic acid or NHS-activated ester is located on the copolymer which can be couple to the QDs.

Organic Compounds

Organic compounds can be produced by living organisms or can be synthesized chemically in the laboratory, and include large polymeric molecules such as proteins, polysaccharides, and nucleic acids as well as small molecules such as primary

metabolites, secondary metabolites, and natural products. In some embodiments, the organic compounds include, but are not limited to, growth factors, proteins, antibodies, DNA, RNA, and peptides. In some embodiments, the organic compound is epidermal growth factor. In some embodiments, the organic compound is a detectable agent.

In some embodiments, the organic compound is a detectable agent such as a fluorescent dye, e.g. Cy series, ALEXA series, BODIPY, Fluorescein, Oregon Green, Rhodamine series, TEXAS RED™ series, coumarins, pyrenes, pyridyloxazole derivatives, naphthalenes, targeting ligands, e.g., biotin, and molecules that can sense environmental change, e.g., pH sensitive, oxygen sensitive, glucose sensitive. These QD conjugates can target specific moieties, e.g., tumors, receptors, proteins, and organs. See e.g. Xiaohu Gao et al. Nature Biotechnology 22: 969-976, 2004 and Howarth, M. et al. Nature Methods 5: 397-399, 2008, the contents of which are incorporated by reference in their entirety. Additionally, QD conjugates can sense their environment, e.g. pH, oxygen, glucose level. See e.g. Somers, R. C. et al. Chem. Soc. Rev. 36: 579-591, 2007 and McLaurin, E. J. et al. J. Am. Chem. Soc. 131 : 12994-13001, 2009, the contents of which are incorporated by reference in their entirety.

Fluorescent Dyes

In some embodiments, dyes can include an NIR contrast agent that fluoresces in the near infrared region of the spectrum. Exemplary near-infrared fluorophores can include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) between about 630 and 1000 nm, e.g., between about 630 and 800 nm, between about 800 and 900 nm, between about 900 and 1000 nm, between about 680 and 750 nm, between about 750 and 800 nm, between about 800 and 850 nm, between about 850 and 900 nm, between about 900 and 950 nm, or between about 950 and 1000 nm. Fluorophores with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm can also be used in the methods described herein. In some embodiments, the fluorescent dye forms a FRET pair with the QD.

Fluorophores useful in the present methods include without limitation: 7-amino-4- methylcoumarin-3 -acetic acid (AMCA), TEXAS RED™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and -6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and -6)- carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3- carboxylic acid, tetramethylrhodamine-5-(and -6)-isothiocyanate, 5 -(and -6)- carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5- (and -6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3- indacenepropionic acid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and

CASCADE™ blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). In some embodiments, fluorescently labeled probes are viewed with a

fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. See, for example, U.S. Pat. No. 5,776,688, which is incorporated by reference in its entirety.

In some embodiments, fluorescent proteins are used and include without limitation: green/yellow/cyan fluorescent protein and variants thereof, and photo- activatable/switchable fluorescent proteins. See e.g. the worldwide web at:

"en.wikipedia.org/wiki/Fluorophore",

"en.wikipedia.org/wiki/Green_fluorescent_protein", and

"en.wikipedia.org/wiki/Photoactivatable_fluorescent_prote in."

In some embodiments, the organic compound is a bio-molecule, e.g., biotin or digoxygenin. Biotin can be detected by avidin conjugated to a detectable marker. In some embodiments, avidin is conjugated to an enzymatic marker such as an alkaline phosphatase or horseradish peroxidase. Enzymatic markers can be detected in standard colorimetric reactions using a substrate and/or a catalyst for the enzyme. Catalysts for alkaline phosphatase can include 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. In some embodiments, diaminobenzoate is used as a substrate for horseradish peroxidase.

Copolymers

The copolymers described herein are useful for coupling to the QDs. Copolymers that are useful for the composition and methods described herein are polymeric imidazole ligands (PILs) that are random copolymers incorporating poly(ethylene) glycol (PEG), amino-PEGn, and imidazole groups for water solubilization, functionalization and QD binding, respectively. For example, a copolymer comprising 30% poly(ethylene) glycol (PEG ₁₂ ), 20%) amino-PEGn, and 50%> imidazole groups can be used. This copolymer has an amino group that acts as a handle for attaching to the activated carboxy group of the norbornene compound. For a description of additional copolymers useful for the compositions and methods described herein, see e.g. WO/2011/022338, which is incorporated by reference in its entirety. In addition to PILs, various copolymers such as thiol based copolymers, e.g. monothiolated copolymers, bidentate thiols, amphiphilic polymers, imidazole based polymer, and functionalized oligomeric phosphine can be used to coat QDs to produce bio-compatible QD samples. See e.g. Medintz, I. L. et al. Nature Materials 4: 435-446, 2005. QDs can phase transfer from hydrophobic growth solutions to water phase after ligand exchange, e.g. coordinating copolymers such as thiol based copolymers and imidazole based polymers, oligomeric phosphines, or encapsulation (amphiphilic polymers) with the copolymers.

Methods of Imaging the Immobilized Target Molecule

The methods described herein can be used to image a bio-molecule of interest in a variety of detection methods suitable for the type of label employed.

The bio-orthogonal conjugates can be detected using detection techniques known in the art. Examples of such techniques include fluorescence detection using instruments such as confocal scanners, confocal microscopes, or CCD-based systems and techniques such as fluorescence, fluorescence polarization (FP), fluorescence resonant energy transfer (FRET), total internal reflection fluorescence (TIRF), and fluorescence correlation spectroscopy (FCS).

In some embodiments, the bio-molecules of interest are fluorescently labeled and then imaged with total internal reflection fluorescence (TIRF) microscopy. For example, fluorescently labeled proteins and nucleic acids can be used to probe bindings at the single molecule level. Thus, exemplary detection methods include radioactive detection, optical absorbance detection, e.g. , UV- visible absorbance detection, optical emission detection, e.g., fluorescence or chemiluminescence. In some embodiments, extended primers are detected on a substrate by scanning all or portions of each substrate simultaneously or serially, depending on the scanning method used. For fluorescence labeling, selected regions on a molecule are serially scanned one-by-one or row-by-row using a fluorescence microscope apparatus. Hybridization patterns can also be scanned using a charge-coupled device (CCD) camera. Methods of Attaching a Functionalized Norbornene to a Copolymer

The norbornene compound is covalently attached to a copolymer through a functional group on the norbornene. In some embodiments, the norbornene compound is functionalized with a -CH ₂ -CO ₂ H group.

In some embodiments, the norbornene compound can be functionalized with a linker group wherein the linker group has a first end and a second end.

The term "linker" as used herein refers to a group of atoms, e.g. 5-100 atoms, and can be comprised of the atoms or groups of atoms, e.g. carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, amide, and carbonyl. The linker is connected at a first end to the norbornene compound through a carbon-carbon bond. In addition, the linker is connected at a second end to the copolymer through a covalent bond. Examples of covalent bonds include, but are not limited to, an amide bond or a triazole ring, through "click chemistry." See, e.g., the Sigma Aldrich catalog and U.S. Patent No. 7,375,234. In some embodiments, the linker is incorporated on the norbornene compound and then the norbornene compound with the incorporated linker i then attach to the copolymer. The copolymer may or may not be coupled to the QD.

As used herein, the term "activated carboxylic acid" refers to a derivative of a carboxyl group that is more susceptible to nucleophilic attack than a free carboxyl group; e.g., acid anhydrides, thioesters, and esters (e.g., an NHS ester).

Synthetic chemistry transformations useful for introducing and reacting such functional groups are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents or Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

All chemicals were purchased from Sigma Aldrich unless noted and were used as received. Selenium shot, cadmium oxide 99.999%, and n-tetradecylphosphonic acid (TDPA) were purchased from Alfa Aesar (Ward Hill, MA). Trioctylphosphine (TOP) and tributylphospine (TBP) were purchased from Strem Chemicals (Buchs, Switzerland). Epithermal Growth Factor (EGF, Human recombinant) and Alexa Fluor® 594 carboxylic acid, succinimidyl ester were purchased from Invitrogen. (lS,2S,4S)-bicyclo[2.2.1]hept- 5-en-2-yl acetic acid was purchased from ChemBridge. All solvents were of reagent grade or higher and were used without further purification.

Tributylphosphine selenide (TBP-Se) was prepared by dissolving 0.15 mmol of selenium shot in 100 mL of TBP under inert atmosphere and stirring vigorously overnight, forming a 1.5 M TBP-Se solution. All air sensitive materials were handled in an Omni-Lab VAC glove box under dry nitrogen atmosphere with oxygen levels < 0.2 ppm. All solvents were spectrophotometric grade and purchased from EMD Biosciences.

Analytical HPLC and LC/MS were performed on a Waters 2695 HPLC equipped with a 2996 diode array detector, a Micromass ZQ4000 ESI-MS module, and a Grace- Vydac RPC18 column (model 218TP5210) at a flow rate of 0.3 mL/min. Preparative HPLC was performed on a Varian ProStar model 210 instrument equipped with a model 335 diode array detector, a model 701 fraction collector, and a Varian RPC18 column (model A6002250X212) at a flow rate of 21 mL/min. All UV/vis spectra were recorded on an Agilent 8453 diode array UV/vis spectrophotometer. Photoluminescence and absorbance spectra were recorded with a BioTek Synergy

4 Microplate Reader. The absorbance of all solutions was kept below 0.1 OD to avoid inner- filter effects. Flash column chromatography was performed on a Teledyne Isco CombiFlash Companion. Polymer molecular weights were determined in DMF solutions on an Agilent 1100 series HPLC/GPC system with three PLgel columns (103, 104, 105 A) in series against narrow polystyrene standards.

Example 1. Synthesis of CdSe (CdS) for QD Preparation

CdS was prepared by placing 0.4 mmol (54.1mg) of CdO, 0.8 mmol (0.2232g) of TDPA, 9.6mmol (3.72g) of TOPO in a 25 mL round bottom flask. The solution was degassed for 1 hour at 160°C and heated to 300°C under argon until CdO dissolved and formed a clear homogenous solution and was followed by pulling a vacuum at 160°C to remove evolved water. The solution was reheated to 360°C under argon and the TBP-Se solution (1.5mL of 1.5M TBP-Se in 1.5mL of TOP) was rapidly added to give the CdSe cores with the first absorption feature at 468 nm. The core was grown at 260°C to produce the core with the desired wavelength.

CdS shells were deposited on CdSe core via a modification of previously reported procedures (Liu, W.et al. Journal of the American Chemical Society 132: 472-483, 2010). Cores isolated by repeated precipitations from hexane with acetone were brought to 180 °C in a solvent mixture of oleylamine (3 mL) and octadecene (6 mL). Aliquots of Cd and

5 precursor solutions were then introduced alternately starting with the metal (Cd) and waiting 15 minutes between the start of each addition. The Cd precursor consisted of 0.33 mmol Cd-oleate and 0.66 mmol oleylamine in a solvent mixture of octadecene (1.5 mL) and TOP (3 mL). The S precursor consisted of 0.3 mmol hexamethyldisilathiane [(TMS) ₂ S] in 6 mL TOP. The dose of each overcoating precursor aliquot corresponded to a single monolayer of ions to the QD surface. Addition of a total of 4 aliquots each of Cd and S yielded QDs with emission at 570 nm and quantum yield close to unity when diluted in octane. A similar procedure was performed on larger CdSe cores to obtain CdSe(CdS) QDs emitting at 605 nm. The extinction coefficient of CdSe(CdS) was calculated using the extinction coefficient of CdSe core from literature (Leatherdale, C. A. et al. J. Phys. Chem. B. 106: 7619-7622, 2002) and assuming that 100% of CdSe cores were retained for the overcoating step.

Example 2. Preparation of Amine- reactive Tetrazine (Tz-NHS)

2,5-dioxopyrrolidin-l-yl 5-(4-(l,2,4,5-tetrazin-3-yl)benzylamino)-5- oxopentanoate (Tz-NHS) was prepared from 3-(4-benzylamino)-l,2,4,5-tetrazine (Tz- benzylamine) that was synthesized as previously described (Devaraj, N. K. et al.

Bioconjugate Chemistry 19: 2297-2299, 2008). Tz-benzylamine (10 mg) was added to a solution of methylene chloride containing 6 mg glutaric anhydride. The solution was stirred overnight at 50°C. The methylene chloride was removed by rotary evaporation and the crude mixture purified by column chromatography resulting in 5-(4-(l, 2,4,5- tetrazin-3-yl)benzylamino)-5-oxopentanoic acid (Tz-acid) in quantitative yield. This acid was then immediately introduced to an acetonitrile (2 mL) solution of N, N'- disuccinimidyl carbonate (68 mg) and triethylamine (30 mg) and allowed to stir at room temperature until the reaction reached completion (monitored by TLC). The acetonitrile was removed by rotary evaporation and the crude mixture purified by column

chromatography yielding 17 mg (80% yield) of the desired Tz-NHS. ¹ HNMR (400 MHz CDC1 ₃ ): δ 10.3-10.2 (s, 1H), 8.7-8.5 (d, 2H), 7.6-7.4 (d, 2H), 6.6-6.2 (br, 1H), 4.7-4.4 (m, 2H), 3.1-2 (m, 10H). LR-MS [M+H]+ calc mass 399.1 found mass 399.2.

Example 3. Synthesis of Norbornene Conjugated Polymeric Imidazole

Copolymers (NB-PIL)

Poly(amino-PEGn) ₂ o _% -PIL was synthesized using a previously reported method (Liu et al). (lS,2S,4S)-bicyclo[2.2.1]hept-5-en-2-yl acetic acid (norbornene) was activated by reacting 0.05 mols of (lS,2S,4S)-bicyclo[2.2.1]hept-5-en-2-yl acetic acid with 0.06 mols n-hydroxysuccinimide (NHS) and 0.06 mol Ν,Ν'- dicyclohexylcarbodiimide (DCC) in anhydrous tetrahydrofuran (THF) for 2 hours at room temperature. NHS activated norbornene was reacted with Poly(amino-PEGn) ₂ o%- PIL in dry THF overnight (2 times excess of norbornene was added to the number of the amine groups in the polymer). After the reaction was completed THF was pulled off and the reaction mixture was redissolved in ethyl acetate to precipitate byproducts.

Precipitates were filtered off using a 0.2 μΜ PTFE syringe filter. Cleanup was repeated several times until no precipitate was observed after pulling off the solvent.

Example 4. Fluorescamine Assay of Amine Reactivity PILs

Stock solutions of amine-containing PIL polymers were made at 20 mg/mL concentration. A serial dilution was made using 1, 2, and 4 uL of polymer stock into 240 uL of PBS buffer, followed by addition of 10 uL of a 30 mg/mL solution of fluorescamine in acetone. This mixture was vortexed and incubated at room temperature for 1 hour before fluorescence analysis on a BioTek plate reader with excitation at 380 nm and detection at 480 nm. The recorded fluorescence intensity signals were calibrated against solutions of known concentrations of methoxyPEGn-NH2.

Example 5. Preparation of Norbornene Coated Water Soluble QDs

Copolymer exchange of native QDs with NB-PIL was performed as described in the previous literature (Liu et al.) To summarize, QDs (1 nmol) were precipitated using hexanes (30 /zL), CHC1 ₃ (30 /zL) and EtOH(200 /zL) and brought into 50 //L of CHC1 ₃ . The QD stock solution was mixed with a solution of NB-PIL (4 mg) in CHC1 ₃ (30 /zL), and stirred for 20 minutes at room temperature, after which 30 of MeOH was added followed by stirring for an additional 20 minutes. QD samples were precipitated by the addition of EtOH (30 /zL), CHC1 ₃ (30 /zL), and excess hexanes. The sample was centrifuged at 4000 g for 2 minutes. The clear supernatant was discarded, and the pellet dried in vacuo, followed by the addition of PBS (500 /zL, pH 7.4). The aqueous sample was then filtered through a 0.2 μπι filter syringe filter before use. Prior to any

conjugation chemistry or cell studies, free copolymer was removed by three cycles of dilution/concentration through an Amicon Ultra Ultracel 50,000 Da MW cutoff filter (Millipore). The quantum yield of norbornene coated QDs was about 70%.

Example 6. Synthesis of 3-(4-benzylamino)-l,2,4,5-tetrazine conjugated to Alexa 594 Alexa Fluor® 594 carboxylic acid, succinimidyl ester (Alexa 594) was reactivated with n-hydroxysuccinimide by adding 1.2 equivalents of NHS and 1.2 equivalents of DCC in dry DMF and reacted for 2 hours at room temperature. 3-(4-benzylamino)- 1 ,2,4,5-tetrazine (1 equivalent) was added to the solution and reacted overnight at room temperature. Completion of the reaction was confirmed using ninhydrin staining.

Example 7. Synthesis of EGF-BAT

Amine reactive tetrazine (4mg/mL) was reactivated with n-hydroxysuccinimide by adding 1.2 equivalents of NHS and 1.2 equivalents of DCC in dry DMF and reacted for 2 hours at room temperature. 50μg of EGF was dissolved in 200/zL IX PBS and 1.2 equivalents of NHS activated tetrazine was added to the solution and reacted overnight at room temperature. The conjugates were dialyzed three times with an Amicon Ultra Ultracel 3,000 Da Mw cutoff filter (Millipore) to get rid of excess NHS, DCC and byproducts.

Example 8. Synthesis of QD-Alexa594 conjugates

200/zL of ~ΙμΜ norbornene coated QDs were mixed with different concentrations of Alexa594 tetrazine in IX PBS and reacted for 4 hours at 37°C. Excess reagents were removed by gel filtration chromatography and dialyzed three times with IX PBS using Amicon Ultra Ultracel 50,000 Da MW cutoff filter. The control experiments were performed using Poly(amino-PEGn)2o _% -PIL coated QDs. Final materials were analyzed by UV-Vis absorption to determine the number of dyes on the QD surface.

Concentrations of QDs and Alexa 594 were measured based on 8d _ye = 90,000 cm ^Λ Μ ^Λ at 590nm for Alexa 594, and 8 _QD = 2,630,000 cm^M ^"1 at 350nm for QD570.

Example 9. Results from Determining Alexa-BAT and QD-Alexa594 Conjugate Efficiencies

The cycloaddition was achieved by functionalizing QDs with norbornene and reacting with BAT-modified substrates. Polymeric imidazole ligands (PILs) were used to prepare norbornene-coated water soluble QDs. PILs are random copolymers incorporating poly(ethylene) glycol (PEG), amino-PEGn, and imidazole groups for water solubilization, functionalization, and QD binding, respectively. The modularity of the polymer and commercial availability of the norbornene allowed for facile incorporation of norbornene groups on the polymer in gram scale. Poly(amino-PEGn) ₂ o _% -PIL (NH ₂ - PIL) was used, which is composed of 30% poly(ethylene) glycol (PEG ₁₂ ), 20% amino- PEGn, and 50%) imidazole groups, and was further modified with n- hydroxysuccinimide(NHS) activated bicyclo[2.2.1]hept-5-en-2-yl acetic acid

(norbornene) via amide coupling (FIG. 1 A). Complete conversion of amines to norbornenes was confirmed by probing free amine in the polymer before and after the conjugation with fluorescamine, an amine-reactive fluorogenic probe (FIG. 2).

Norbornene-coated QDs were prepared by ligand exchange of natively capped QDs with the norbornene modified PIL (NB-PIL) (FIG. IB).

To determine conjugation efficiencies of the cycloaddition on QDs, norbornene- coated QDs were conjugated with BAT modified Alexa594 (Alexa-BAT) (FIG. 1B-D). Coupling yields were determined through knowledge of the extinction coefficients of the dye and QDs and measurement of the product absorption spectra. The number of Alexa dyes conjugated to the norbornene coated QDs varied depending on the excess of Alexa- BAT (FIG. 1C-D). Increasing the dye concentration to lOOx excess, led to a saturation coupling yield of -16 dyes/QD (shown in Table 1 below). This number effectively represented the average number of reactive norbornene molecules on the surface of each QD. The number of coupled dyes can be increased by further increasing the composition of norbornenes in the starting polymer.

Table 1. QD-Alexa 594 Conjugation Ratios

Mixed QD QD Dye Dye Purified

Dye:QD Dye:QD

Ratio Abs @ 350 Cone (μΜ) Abs @ 590 Cone (μΜ) Ratio

10 0.25 1 0.(W54 0.035 0.3SS 4.072

40 0.425 0.1616 0.165 1.83 1 1.336

100 0.4 1 0. 1 639 0.245 2.72 1 6.609

200 0.442 0.1681 0.232 2.58 15.373

Example 10. Synthesis of QD-EGF Coupling

Norbornene coated QDs (0.2 nmol) and 0.4 nmol of EGF-tetrazine were mixed in IX PBS with the final concentration of ΙμΜ for QDs and incubated for 2 hours at 37°C. Unreacted EGF was removed by three cycles of dilution/concentration through an Amicon Ultra Ultracel 50,000 Da MW cutoff filter (Millipore).

Example 11. Quantum yield (QY) of QDs

The QY of QD570 was measured relative to Rhodamine 610 (QY 68% in ethanol) with excitation at 505 nm and QY of QD605 was measured relative to Rhodamine 640 (QY 100% in ethanol with excitation at 535nm). Solutions of QDs in octane (native CdSe/CdS QDs) or PBS (QDs after ligand exchanged with either Poly(amino-PEGn)2o%- PIL or NB-PIL) and dye in ethanol were optically matched at the excitation wavelength. Fluorescence spectra of QD and dye were taken under identical spectrometer conditions in quadruplicate and averaged. The optical density was kept below 0.1 at the _max , and the integrated intensities of the emission spectra, corrected for differences in index of refraction and concentration, were used to calculate the quantum yields using the expression QYQD = (Absorbance)d _ye /(Absorbance)QD x (Peak Area)oD /(Peak Area) _Dye

(HQD solvent) ² /(riDye solvent) ² X QYDye-

Example 12. Gel Filtration Chromatography (GFC) GFC was performed using an AKTAprime Plus chromatography system from Amersham Biosciences equipped with a self-packed Superdex 200 10/100 column. PBS (pH 7.4) was used as the mobile phase with a flow rate of 1.0 mL/min. Detection was achieved by measuring the absorption at 280 nm.

Example 13. Transmission Electron Microscopy

The inorganic size of CdSe(CdS) QDs was determined to be approximately 4.6 nm using a JEOL 200CX TEM operating at 200 kV (FIG 3). One drop of a dilute sample of QDs in hexane precipitated two times using acetone was placed onto a Formvar coated copper grid, allowed to settle for 20 seconds, and wicked away using an absorbent tissue. Size analysis was performed on captured digital images using ImageJ 1.34s.

Example 14. Cell culture and labeling

A-431 human epidermoid carcinoma cells were grown in DMEM (Invitrogen) with 10% Fetal Bovine Serum (Invitrogen), 50 μg /mL penicillin and 50 μg /mL streptomycin (Invitrogen). When labeling cells with preformed QD-EGF conjugates (FIG. 4B), cells were rinsed with 4°C 1% Bovine Serum Albumin (BSA) in PBS and incubated with 50 nM QD-EGF conjugates at 4°C for 30 minutes. For in situ click conjugation between tetrazine and norbornene on cells (FIG. 4C), cells were rinsed with 4°C 1% BSA in PBS, incubated with 200 nM EGF-BAT at 4°C for 30 minutes, and then rinsed three times with 1% BSA in PBS to block non-specific binding. Subsequently, norbornene coated QDs at varying concentrations were added to the cells and incubated for 30 minutes at 37°C. The cells were then washed three times with 25°C PBS to remove excess QDs.

Example 15. Fluorescence imaging

Cells were imaged with an epifluorescence microscope (Nikon) with a 60x water- immersion objective and Princeton instruments MicroMax Camera with a 1.5x magnification tube lens. Bright field images were collected using differential interference contrast with an exposure time of 100 ms and fluorescence images were collected by exciting with a 488 nm Argon-ion laser line combined with a D605/30M emission filter. Exposure times for fluorescence imaging were 200 ms for QD blinking time-lapse imaging and 500 ms for all others. All fluorescence image frames for the QD blinking video were background corrected using Matlab.

Example 16. Results from Coupling QDs to EGF

To illustrate the utility of the coupling chemistry for live cell imaging with QDs, epidermal growth factor receptors (EGFRs) were labeled which are abundant on the surface of human skin cancer cells. Cellular labeling was achieved either directly through use of pre-formed QD-EGF conjugates (FIG. 4B) or by performing in situ conjugation of the norbornene-coated QDs to BAT modified EGFs on the cell surface (FIG. 4C). For direct labeling, the norbornene coated QDs were coupled with BAT modified EGF (FIG. 4A) and 50 nM of the resulting QD-EGF conjugates were added to A-431 human epidermoid carcinoma cells at 4 °C for 30 minutes (FIG. 5B). For in situ conjugation, cells were incubated with 200 nM BAT modified EGF (BAT-EGF) at 4 °C for 30 minutes and labeled with 800 nM of norbornene coated QD at 37 °C for 30 minutes (FIG. 5D). Control experiments using the same procedures but with QDs coated with poly(PEGi ₂ )-PIL, composed of 50 % imidazole and 50% PEGi ₂ (without norbornene), are shown in FIGs. 5 A and 5C.

As FIGs. 5A-C demonstrate, successful labeling was achieved using either labeling technique. The fast rate of the coupling reaction in serum allowed for in situ conjugation of norbornene-coated QDs to BAT-EGF labeled cells. In addition, this method did not result in an increased QD size and in general worked on cells with endogenously expressed receptors.