Click Chemistry Introduction

Transcription

1 Introduction 'Click chemistry' was defined by Sharpless and co-workers in 2001 (1) to describe a set of powerful, highly reliable and selective organic reactions which can be used for the rapid and facile synthesis of useful new compounds and combinatorial libraries. Each of these compounds is composed of small, modular sub-units stiched together through heteroatom linkages (C-X-C). Click chemistry reactions are simple, modular, stereospecific, very high yielding, wide in scope, can be conducted in benign/easily removable solvents, and generate side products which are easily removable by non-chromatographic methods. The primary driving force behind the development of click chemistry is the pharmaceutical industry's need to generate very large combinatorial libraries of small-molecule (< 500 Dalton) compounds that can be screened as drug candidates. Click chemistry has the potential to accelerate the drug discovery process, as it makes each reaction in the multi-step synthesis of a small molecule fast, efficient and predictable. Although there are several types of organic reactions that fit the definition of click chemistry, for modification of oligonucleotides, the relevant one is the copper(i)-catalyzed [3+2] cycloaddition reaction between alkynes and azides (1,2). This reaction is extremely selective and regiospecific for conjugation reactions involving an oligo and a labeling moiety, as well as coupling reactions between two oligos. More detailed descriptions of specific click chemistry applications are provided in ' Applications'. Page 1

2 Design Protocols This section contains an example of a Copper(I)-catalyzed click reaction. This protocol may be used as a starting point for optimization of your particular click chemistry procedures. I. Preparation of the 'Click Solution' 1. NOTE: The 'click solution' (0.1 M CuBr / 0.1 M TBTA 1:2 (v/v) in DMSO/t-BuOH 3:1 (v/v)) must always be freshly prepared prior to use! 2.Dissolve 1 mg CuBr in 70 μl DMSO/t-BuOH 3:1 (v/v) to obtain a 0.1 M solution. This solution must be freshly prepared and cannot be stored. 3.Dissolve 54 mg TBTA in 1 ml DMSO/t-BuOH 3:1 (v/v) for a 0.1 M solution. This solution can be stored at -20C. 4.Add 1 volume of the 0.1 M CuBr solution quickly to 2 volumes of the 0.1 M TBTA solution to obtain the click solution, which is ready to use. II. Click Procedure for Short DNA Oligos Procedure using CuBr: To 5 μl of a 2 mm DNA solution (10 nmol) in water, 2 μl of an azide solution (50 mm, 50 nmol, 5 eq. in DMSO or in 3:1 (v/v) DMSO/t-BuOH), 3 μl of a freshly prepared solution containing 0.1 M CuBr and 0.1 M TBTA ligand in a 1:2 (v/v) ratio in 3:1 (v/v) DMSO/t-BuOH is added. The mixture is thoroughly mixed and shaken at 25C for 3 h. The reaction is subsequently diluted with 0.3 M NaOAc (100 μl) and the DNA is precipitated using 1 ml cold EtOH. The supernatant is then removed and the residue is washed twice with 1 ml cold EtOH. The washed residue is re-dissolved in pure water (20 μl) and can be used without further purification. Page 2

3 Applications Although the copper(i)-catalyzed alkyne-azide [3+2] cycloaddition reaction has many potential uses as a method for synthesis of unique oligo-based research tools, two specific applications currently dominate, (A) conjugation of anti-sense/sirna oligonucleotides to cell-penetrating peptides (CPP), and (B) labeling of oligonucleotides with biotin and/or fluorescent dyes. The efficacy of anti-sense and sirna oligos in vivo is severely limited due to their inability to cross the cell membrane (3). One method for significantly increasing cell uptake of these oligos is to covalently conjugate cell penetrating peptides (CPP) to the oligo, most commonly through amide or disulfide linkages (4,5). While relatively straightforward to perform, such conjugations show wide variability in final yield, and often require complex and/or multiple purification. In addition, if the peptide is highly cationic, achieving a successful conjugation reaction can itself be problematic, due to non-specific binding between the cationic peptide and the anionic phosphate backbone of the oligo (6). Using click chemistry to form the CPP-oligo conjugate by linking an azidopeptide to an alkyne-modified oligo can provide an effective solution to these problems, as yield is essentially quantitative, and because the conjugation can be performed on a solid support, the need for expensive, multi-step purification is often eliminated (7-9). Please see the references provided for more details. Similar advantages (quantitative yield, simple purification) favor the use of click chemistry for labeling oligos with such moieties as biotin, fluorescent dyes, and haptens (10-12). In all these cases, the oligo is alkyne-modified and the labels all contain an active azide group. A number of azide-modified labels are available, including biotin, desthiobiotin, 6-FAM, HEX and TET, among others. Additional labels will become available over time. Page 3

4 References 1. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Akhtar, S., Hughes, M.D., Khan, A., Bibby, M., Hussain, M., Nawaz, Q., Double, J., Sayyed, P. The delivery of antisense therapeutics. Adv. Drug. Deliv. Rev. (2000), 44: Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, A., El Andaloussi, S. Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug. Chem. (2008), 19: Frederic, H., May Catherine, M., Gilles, D., Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. (2009), 157: Lu, K., Duan, Q-P., Ma, L., Zhao, D-X. Chemical strategies for the synthesis of peptide-oligonucleotide conjugates. Bioconjug. Chem. (2010), 21: Gogoi, K., Mane, M.V., Kunte, S.S., Vaijayanti, A.K. A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water. Nucleic Acids Res., (2007), 35: e Brown, S.D., Graham, D. Conjugation of an oligonucleotide to Tat, a cell-penetrating peptide, via click chemistry. Tet. Lett., (2010), 51: Wenska, M., Alvira, M., Steunenberg, P., Stenberg, A., Murtola, M., Stromberg, R. An activated triple bond linker enables click attachment of peptides to oligonucleotides on solid support. Nucleic Acids Res., (2011), 39: Seo, T.S., Li, Z., Ruparel, H., Ju, J. Click chemistry to construct fluorescent oligonucleotides for DNA sequencing. J. Org. Chem., (2003), 68: Hall, L.M., Gerowska, M., Brown, T. A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers. Nucleic Acids Res., (2012), 40: e Wengel, J., Astakhova, I.K. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes. Chemistry, (2013), 19: Page 4

5 Modification Catalog Number Alkyne C8 dc (5-Octadiynyl-dC) Alkyne C8 dt (5-Octadiynyl-dT) Alkyne-Modifier Serinol Alkyne- C2 NHS Azide C Azide C6 (5') Azide dt (5') Azide-C2 NHS Azide butyrate NHS BiotinTEG Azide Coumarin Azide DBCO-C2 NHS DBCO-dT DBCO-sulfo-NHS DBCO-TEG (5') DesthiobiotinTEG Azide FAM-TEG Azide Halotag Conjugation Bromohexyl (5') HEX-Azide Hexynyl (Alkyne) Modifier (5') Page 45

6 Iodo-dT Propargyl-5-Me-dC(3') TET Azide Page 56

7 Alkyne C8 dc AlkyneC8dC Reference Catalog Number Y Molecular Weight(mw) This modification is on HOLD pending licensing arrangements with baseclick GmbH Alkyne C8 dc and dt can be used to incorporate an active alkyne onto the 5 - or 3 -end of an oligonucleotide, as well as at an internal position. The alkyne group is separated from the oligo by an 8 carbon spacer arm, which serves to reduce steric interaction between the reactive group and the oligo and allows multiple sites to be used. The presence of the alkyne allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate it to a variety of azide-containing labels/tags (e.g., fluorescent dyes, biotin, or oligos, with extremely high regioselectivity and efficiency (1,2). When conjugation to an azide-oligo is desired, preparation of the azide-oligo can be achieved using either an Azidobutyrate NHS Ester or the 5 -Bromohexyl modifier (see their respective tech sheets for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 7

8 Alkyne C8 dt AlkyneC8dT Reference Catalog Number Y Molecular Weight(mw) This modification is on HOLD pending licensing arrangements with baseclick GmbH Alkyne C8 dt and dc can be used to incorporate an active alkyne onto the 5 - or 3 -end of an oligonucleotide, as well as at an internal position. The alkyne group is separated from the oligo by an 8 carbon spacer arm, which serves to reduce steric interaction between the reactive group and the oligo and allows multiple sites to be used. The presence of the alkyne allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate it to a variety of azide-containing labels/tags (e.g., fluorescent dyes, biotin, or oligos, with extremely high regioselectivity and efficiency (1,2). When conjugation to an azide-oligo is desired, preparation of the azide-oligo can be achieved using either an Azidobutyrate NHS Ester or the 5 -Bromohexyl modifier (see their respective tech sheets for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 8

9 Alkyne- Serinol Alkyne Ser Reference Catalog Number Y Molecular Weight(mw) Alkyne Modifier Serinol can be used to incorporate an active alkyne onto the 5 - or 3 -end of an oligonucleotide, as well as at an internal position. The alkyne group is separated from the oligo by an 11-atom spacer arm, which serves to reduce steric interaction between the reactive group and the oligo. The presence of the alkyne allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate it to a variety of azide-containing labels/tags (e.g., fluorescent dyes, biotin, or oligos, with extremely high regioselectivity and efficiency (1,2). When conjugation to an azide-oligo is desired, preparation of the azide-oligo can be achieved using either an Azidobutyrate NHS Ester or the 5 -Bromohexyl modifier (see their respective tech sheets for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 9

10 Alkyne-C2 NHS Alkyne-C2 N Reference Catalog Number Y Molecular Weight(mw) Alkyne NHS modification is a post synthesis conjugation to a primary amino group. The amino group can be placed at the 5' and 3' and for internal positions an amino modified base is used, e.g Amino dt C6 Alkyne NHS ester can be used to incorporate an active alkyne onto the 5 - or 3 -end of an oligonucleotide, as well as at an internal position. Incorporation of this modification to the oligo is done via conjugation to an active primary amine (such as Amino Linker C6). As a result, the alkyne group is separated from the oligo by a spacer arm of varying length, which serves to reduce steric interaction between the reactive group and the oligo. The presence of the alkyne allows the user to use Click Chemistry (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate it to a variety of azide-containing labels/tags (e.g., fluorescent dyes, biotin, or oligos, with extremely high regioselectivity and efficiency (1,2). When conjugation to an azide-oligo is desired, preparation of the azide-oligo can be achieved using either an Azidobutyrate NHS Ester or the 5 -Bromohexyl modifier (see their respective tech sheets for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 10

11 Azide C3 3' N3-C3 Reference Catalog Number Prime N N Molecular Weight(mw) Copper-free Modifications Use azide modified oligos with DBCO Cyclooctyne-based modifications for ease of copper-free click reagents. These are simple to use and has excellent click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature. Azide C3 is available to introduce a stable azide group at the 3' of an oligo. Use Azide butyrate NHS [ ] for introduction of azide at internal or 5' position by conjugating to an amino-modified oligonucleotide. Introduction can be done at either the 5'- or 3'-end, or internally. To do this, the oligo first must be synthesized with a primary amino functional group modification, e.g Amino C6 for the 5' end or amino C7 for the 3' end for the ends) or the amino C6 version of the base phosphoramidite (for internal labeling). The Azidobutyrate NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The presence of the azide allows the user to use "" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2]: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes pdf Print Date Version : February 19, 2019 Page 12

12 Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 12

13 Azide C6 (5') N3-C6 Reference Catalog Number Prime N N Molecular Weight(mw) Copper-free Modifications Use azide modified oligos with DBCO Cyclooctyne-based modifications for ease of copper-free click reagents. These are simple to use and has excellent click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature. Azide C3 is available to introduce a stable azide group at the 3' of an oligo. Use Azide butyrate NHS [ ] for introduction of azide at internal or 5' position by conjugating to an amino-modified oligonucleotide. Introduction can be done at either the 5'- or 3'-end, or internally. To do this, the oligo first must be synthesized with a primary amino functional group modification, e.g Amino C6 for the 5' end or amino C7 for the 3' end for the ends) or the amino C6 version of the base phosphoramidite (for internal labeling). The Azidobutyrate NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The presence of the azide allows the user to use "" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2]: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes pdf Print Date Version : February 19, 2019 Page 14

14 Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 14

15 Azide dt (5') N3-dT Reference Catalog Number Prime N N Molecular Weight(mw) Copper-free Modifications Use azide modified oligos with DBCO Cyclooctyne-based modifications for ease of copper-free click reagents. These are simple to use and has excellent click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature. Azide C3 is available to introduce a stable azide group at the 3' of an oligo. Use Azide butyrate NHS [ ] for introduction of azide at internal or 5' position by conjugating to an amino-modified oligonucleotide. Introduction can be done at either the 5'- or 3'-end, or internally. To do this, the oligo first must be synthesized with a primary amino functional group modification, e.g Amino C6 for the 5' end or amino C7 for the 3' end for the ends) or the amino C6 version of the base phosphoramidite (for internal labeling). The Azidobutyrate NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The presence of the azide allows the user to use "" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2]: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes pdf Print Date Version : February 19, 2019 Page 16

16 Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 16

17 Azide-C2 NHS N3-C2 N Reference Catalog Number Y Molecular Weight(mw) 85 Azide C2 NHS ester can be used to introduce an active azide group to an amino-modified oligonucleotide. Introduction can be done at either the 5'- or 3'-end, or internally. To do this, the oligo first must be synthesized with a primary amino functional group modification, e.g Amino C3, C6 or C12 for the 5' end or amino C3, C6 or C7 for the 3' end for the ends) or the amino C6 version of the base phosphoramidite (for internal labeling). The Azide C2 NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The presence of the azide allows the user to use "Click Chemistry" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 17

18 Azide-C4 NHS (butyrate) N3-C4 N Reference Catalog Number Y Molecular Weight(mw) Azidobutyrate NHS ester can be used to introduce an active azide group to an amino-modified oligonucleotide. Introduction can be done at either the 5'- or 3'-end, or internally. To do this, the oligo first must be synthesized with a primary amino functional group modification, e.g Amino C6 for the 5' end or amino C7 for the 3' end for the ends) or the amino C6 version of the base phosphoramidite (for internal labeling). The Azidobutyrate NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The presence of the azide allows the user to use "" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 18

19 BiotinTEG Azide Bio-TEG-N3 Reference Catalog Number Y Molecular Weight(mw) Biotin-TEG Azide is a biotin attached to a 15-atom mixed polarity triethylene glycol spacer with an azide group at the end of the spacer. The presence of the azide allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the Biotin-TEG Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). The spacer acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin for capture and immobilization of the oligo. Additional technical details for biotin are presented in the Biotin technical sheet. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: pdf Print Date Version : February 19, 2019 Page 19

20 Coumarin Azide Cou-N3 Reference Catalog Number Y Molecular Weight(mw) Coumarin (7-Hydroxycoumarin)-Azide is a fluorescent dye containing a terminal azide group. Coumarin is also known as umbelliferone. Coumarin is highly fluorescent and ph-sensitive, with an absorbance maximum of 358 nm and an emission maximum of 480 nm; thus it emits in the blue region of the visible spectrum. The presence of the azide allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the Coumarin-Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). Because coumarin is effectively quenched if its hydroxyl group is either alkylated or phosphorylated, it is useful in high-throughput screening for enzyme lipases and phosphatases. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: pdf Print Date Version : February 19, 2019 Page 20

21 DBCO-C2 NHS DBCO-C2 N Reference Catalog Number Y Molecular Weight(mw) 288 Copper-free Modifications Cyclooctyne-based modifications offers the ease of copper-free click reagents. These are simple to use and has excellenet click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature. Addition of DBCO-C2 NHS is post synthesis and requires synthesis of oligo with primary amino group pdf Print Date Version : February 19, 2019 Page 21

22 DBCO-dT DBCO-dT Reference Catalog Number Y Molecular Weight(mw) DBCO-DT and DBCO-TEG modification has a setup charge of $ per order for special synthesis reagents. Dibenzocyclooctyne Group (DBCO)Copper-free Modifications DBCO conjugation chemistry is based on the reaction of a dibenzylcyclooctyne (DBCO) linker with an azide linker to form a stable triazole. The dibenzocyclooctyne group (DBCO) allows Copper-free to be done with live cells, whole organisms, and non-living samples. DBCO groups will preferentially and spontaneously label molecules containing azide groups ( N3). Within physiological temperature and ph ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. Reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group ( SH, thiol). Cyclooctyne-based modifications offers the ease of copper-free click reagents. These are simple to use and has excellent click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature pdf Print Date Version : February 19, 2019 Page 22

23 DBCO-sulfo-NHS DBCO-S-NHS Reference Catalog Number Y Molecular Weight(mw) Copper-free Modifications Cyclooctyne-based modifications offers the ease of copper-free click reagents. These are simple to use and has excellenet click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature. Addition of DBCO-Sulfo-NHS is post synthesis and requires synthesis of oligo with primary amino group pdf Print Date Version : February 19, 2019 Page 23

24 DBCO-TEG (5') DBCO-TEG Reference Catalog Number Prime N N Molecular Weight(mw) DBCO-DT and DBCO-TEG modification has a setup charge of $ per order for special synthesis reagents. Copper-free Modifications Cyclooctyne-based modifications offers the ease of copper-free click reagents. These are simple to use and has excellenet click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5 -DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature pdf Print Date Version : February 19, 2019 Page 24

25 DesthiobiotinTEG Azide DesBioTEG-N3 Reference Catalog Number Y Molecular Weight(mw) Desthiobiotin-TEG Azide is a desthiobiotin attached to a 15-atom mixed polarity triethylene glycol spacer with an azide group at the end. The presence of the azide allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the Desthiobiotin-TEG Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). The spacer acts to minimize steric hindrance between the desthiobiotin moiety and the oligo. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (3). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (4). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (5). Gene Link recommends substitution of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: pdf Print Date Version : February 19, 2019 Page 26

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27 FAM-TEG Azide FAM-TEG-N3 Reference Catalog Number Y Molecular Weight(mw) FAM (6-carboxyfluorescein)-TEG Azide is a 6-FAM fluorescent dye attached to a 15-atom mixed polarity triethylene glycol spacer with an azide group at the end of the spacer. 6-FAM is the most commonly used fluorescent dye for labeling oligonucleotides, and is reactive and water-soluble, with an absorbance maximum of 492 nm and an emission maximum of 517 nm. The presence of the azide allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the 6-FAM-TEG Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). The spacer acts to minimize steric hindrance between the biotin moiety and the oligo. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: pdf Print Date Version : February 19, 2019 Page 27

28 Halotag Conjugation Bromohexyl (5') BrHexyl Reference Catalog Number Prime N N Molecular Weight(mw) Bromohexyl modification is available as an Azide for see Azide C6 [ ] and as bromohexyl for Halotag protein conjugation. For Halotag protein conjugation a Spacer 18 modification should be added internally next to the 5'-Bromohexyl. Additional charge applies for Spacer 18 modification. Bromohexyl modification version has a setup charge of $ for mild synthesis reagents per order. Halotag Protein Oligo Conjugation Click here for a validated Glen Research Protocol for Oligo Conjugation to Halo Tagged Protein The strategy of small-molecule fluorescent labeling of genetically encoded proteins has become a popular alternative to GFP labeling. Among the most widely used approaches is the HaloTag method developed by Promega, which utilizes a bacterial haloalkane dehalogenase. The enzyme removes halides from aliphatic hydrocarbons by a nucleophilic displacement mechanism to form a covalent ester linkage between the haloalkane and Asp106 in the enzyme. In the wild type haloalkane dehalogenase, the ester is quickly hydrolyzed by histidine 272 in the catalytic active site. However, by mutating the histidine to phenylalanine, the HaloTag variant renders the covalent ester bond stable toward hydrolysis. Oligonucleotides should be synthesized with Bromohexyl at the 5' end with an adjacent internal Spacer 18 followed by the sequence of choice to be conjugated. Please note that for our online ordering system the addition of Spacer 18 modification is not automatic and should be added as an internal modification. For fluorescent detection the oligo can be labelled at the 3' end with a fluorophore. Halotag Protein Conjugation Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol., 2008, 3, Vijay Singh, Shenliang Wang, and Eric T. Kool, Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone. J. Am. Chem. Soc., 2013, 16, pdf Print Date Version : February 19, 2019 Page 28

29 HEX-Azide-6 HEX-N3 Reference Catalog Number Y Molecular Weight(mw) HEX (Hexachloro-fluorescein)-Azide is a fluorescent dye containing an terminal azide group. HEX has an absorbance maximum of 535 nm and an emission maximum of 556 nm. The presence of the azide allows the user to use "Click Chemistry" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the HEX-Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5'-Hexynyl (Alkyne) modifier or for copper free conjugation use the cyclooctyne DBCO dt, DBCO TEG(see its respective tech sheet for details). References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: pdf Print Date Version : February 19, 2019 Page 29

30 Hexynyl (Alkyne) Modifier (5') 5Hexynyl Reference Catalog Number Prime N N Molecular Weight(mw) The 5 -Hexynyl modifier can be used to incorporate an active alkyne onto the 5 -end of an oligonucleotide. The alkyne is separated from the 5 -end nucleotide base by a 4-carbon spacer arm, which serves to reduce steric interaction between the reactive group and the oligo. The presence of the alkyne allows the user to use (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate it to a variety of azide-containing labels/tags (e.g., fluorescent dyes, biotin), or oligos, with extremely high regioselectivity and efficiency (1,2). When conjugation to an azide-oligo is desired, preparation of the azide-oligo can be achieved using either an Azidobutyrate NHS Ester or the 5 -Bromohexyl modifier (see their respective tech sheets for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 30

31 Iodo-dT 5'-Az-dT Reference Catalog Number Y Molecular Weight(mw) '-Iodo dt modification is available as an Azide dt [Catalog #: ] for. 5'-Iodo dt modification has a setup charge of $ for mild synthesis reagents per order. Azide version does not have additional charges Iodo-dT (5') can be used to introduce an active azide group to the 5'-end of an oligonucleotide. The oligo is chemically synthesized with Iodo-dT at the 5' end and then post synthesis converted to an active azide. The oligo is provided as an azide or 5'-iodo. The selection should be indicated in the comments of the order. The presence of the azide allows the user to use "" (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the azide-modified oligo to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5'-Hexynyl modifier (see its respective tech sheet for details). Click chemistry can be used to form short, cyclic oligos that can be used as research tools in various biophysical and biological studies (3). In particular, they have considerable potential for in vivo work, as cyclic oligos are known to be very stable in serum for up to several days. References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: Kumar, R., El-Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using. J. Am. Chem. Soc. (2007), 129: pdf Print Date Version : February 19, 2019 Page 31

32 Propargyl-5-Me-dC(3') Pro-5me-dC Reference Catalog Number Prime N N Molecular Weight(mw) Propargyl refers to triple/alkyne bond structure next to a saturated position with the following structure HC C CH2,. Placing a propargyl group at the 3' end in conjunction with an azide at the 5' position can be ligated using click chemistry. Ligation of an oligo containing a 5'-azide with an oligo containing a 3'-propargyl group using leads to a triazole linkage that has been shown to have in vivo biocompatibility. This technique has been used to synthesize DNA constructs up to 300 bases in length. When the resultant triazole linkage was placed in a PCR template, various polymerases were able to copy the sequence correctly. The linkage has also been shown to be compatible with transcription and rolling circle amplification, as well as gene expression in E. coli. In the RNA world, a hammerhead ribozyme containing the triazole linkage at the substrate cleavage site has been shown to retain its activity. A large variety of applications is envisaged for this biocompatible chemical ligation. An azide can be introduced at the 5' end of an oligo using Iodo-dT (5'); catalog number pdf Print Date Version : February 19, 2019 Page 32

33 TET-Azide TET-N3 Reference Catalog Number Y Molecular Weight(mw) TET (Tetrachloro fluorescein)-azide is a fluorescent dye containing a terminal azide group. TET has an absorbance maximum of 522 nm and an emission maximum of 538 nm. The presence of the azide allows the user to use Click Chemistry (a [3+2] cycloaddition reaction between alkynes and azides, using copper (I) iodide as a catalyst) to conjugate the TET-Azide to a terminal alkyne-modified oligo with extremely high regioselectivity and efficiency (1,2). Preparation of the alkyne-modified oligo can be achieved using the 5 -Hexynyl modifier (see its respective tech sheet for details). References 1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: pdf Print Date Version : February 19, 2019 Page 33