Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction

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Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition

reaction

Dissertation

zur Erlangung des Akademischen Grades des Doktors der Naturwissenschaften

(Dr. rer. nat.)

an der Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Chemie

vorgelegt von

Samuel Hieronymus Weisbrod

Tag der mündlichen Prüfung: 15. Juni 2010 1. Referent: Prof. Andreas Marx

2. Referent: Prof. Valentin Wittmann

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Publications

Part of this work has been published:

I. Journals

1. S. H. Weisbrod, A. Marx. A nucleoside triphosphate for site-specific labelling of DNA by the Staudinger ligation, Chem. Commun. 2007, 1828–1830.

2. A. Baccaro, S. H. Weisbrod, A. Marx. DNA conjugation by the Staudinger ligation: new thymidine analogues,Synthesis 2007, 1949–1954.

3. S. H. Weisbrod, A. Marx. Novel strategies for the site-specific covalent la- belling of Nucleic Acids,Chem. Commun. 2008, 5675–5685.

4. S. H. Weisbrod, A. Marx, Synthesis of water soluble phosphinophenol for traceless Staudinger ligation,Synlett 2010, 787–789.

II. Book chapter

1. S. H. Weisbrod, A. Baccaro, A. Marx. Site-specific DNA labelling by Staudinger ligation,Methods in Molecular Biology 2011,751, 195–207.

III. Conference contribution

1. S. H. Weisbrod, A. Baccaro, A. Marx. DNA Conjugation by Staudinger Liga- tion Nucleic Acids Symp. Ser. 2008,52, 383–384.

Publications related to this work:

I. Journals

1. S. Liu, S. H. Weisbrod, Z. Tang, A. Marx, E. Scheer, A. Erbe. Direct measure- ment of electrical transport through G-quadruplex DNA with mechani- cally controllable break junction electrodes, Angew. Chem. Int. Ed. 2010, 49, 3313–3316.

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Danksagung

Die vorliegende Arbeit entstand in der Zeit von Mai 2006 bis März 2010 im Arbeitskreis von Herrn Prof. Andreas Marx am Lehrstuhl für Organische und Zelluläre Chemie im Fachbereich Chemie der Universität Konstanz.

Mein besonderer Dank gilt Herrn Prof. Andreas Marx für die Überlassung des sehr interessanten und herausfordernden Themas sowie für das in mich gesetzte Vertrauen, das viel Raum für die selbstständige Bearbeitung und kreative Gestaltung erlaubte. Die exzellenten Arbeitsbedingungen und Laborausstattung haben ebenso zum Gelingen dieser Arbeit beigetragen.

Bedanken möchte ich mich auch bei Herrn Prof. Valentin Wittmann für die Übernahme des Zweitgutachten und Herrn Prof. Stefan Mecking für die Übernahme des Prüfungsvor- sitzes.

Weiterhin möchte ich mich bei allen jetzigen und früheren Mitgliedern der Arbeitsgruppe für die unterhaltsame Zeit, gute Arbeitsatmosphäre und Hilfsbereitschaft bedanken. Beson- ders bedanken möchte ich mich bei meinen langjährigen Laborkolleginnen Anna Baccaro und Anke Gerull für die unkomplizierte Zusammenarbeit. Weiterhin danke ich allen Mi- tarbeiterpraktikanten und wissenschaftlichen Hilfskräften, die mit ihrer Mitarbeit meine Forschungen beschleunigt haben. Ebenso möchte ich mich bei Anke Friemel und Ulrich Haunz bedanken für ihre Unterstützung bei der Durchführung von NMR-Experimenten.

Meinen Lektoren Bastian Holzberger und Anna Baccaro gilt mein Dank für die Durch- sicht meiner Arbeit.

Bei der DFG (SPP1243) möchte ich mich für die teilweise Finanzierung meiner Arbeit bedanken.

All meinen Freunden und Bekannten im Fachbereich Chemie danke ich für die vielen geselligen Stunden und den wissenschaftlichen Austausch, besonders danke ich der Fach- schaft Chemie für die zahlreichen Stunden während der Mittagspause, dem Chemikerkicken und den "Ersti"hütten.

Für die Ablenkung von chemischen Fragestellungen danke ich meiner Skatrunde und allen Mitgliedern der Salsatanzgruppe Rumba Rica.

Meinen Eltern danke ich für die liebevolle Unterstützung in allen Lebenslagen.

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1 Introduction

1.1 Relevance and applications of labelled DNA

Deoxyribonucleic acid (DNA) is a biopolymer consisting of four different monomeric units (nucleotides). In cells DNA is responsible for the information storage in which the sequence of the different nucleobases code for the proteins. The copying and translation of the information is based upon the molecular recognition of Watson - Crick base pairing and the ability of nucleic acids to form double stranded regions. Watson - Crick base pairing is specific and easily predictable and the resulting double stranded DNA (dsDNA) of complementary strands form geometrically well-defined duplex structures. The same properties that allow efficient storage and replication of the genetic code facilitate the self-assembly of simple oligodeoxynucleotides (ODNs). This basic principle has been used for the construction of two- and three-dimensional nanostructures^1–6 and is the basis for ODN markers in many diagnostic applications^{7, 8} as well as DNA mediated reactions or catalysts.^9–11

In nanotechnology the DNA scaffold has to be equipped with functional molecules that provide e.g. new electrical^{12, 13} magnetic^{12, 14} or light transporting properties¹⁵for the construction of nanowires or proteins for artificial multi-enzymes.^{16, 17} For diagnostic applications conjugation of DNA to fluorescent dyes,^{18, 19} affinity tags like biotin or antibodies, proteins, carbohydrates²⁰ and chip surfaces²¹ is needed, whereas for DNA programmed synthesis reactants or catalysts have to be bound to DNA.^22–25In search of highly specific aptamers or DNA based catalysts^{26, 27} the extension of DNA binding properties beyond simple H-bonding capabilities and polyanionic character is highly desirable. In any case the functionalization of the DNA is of capital importance.

There are several strategies to synthesize labelled DNA strands. Short highly modified DNA strands are accessible by solid-phase synthesis. If the incorporation of the label can be accomplished within the synthesizer cycle, fewer steps and only a single purification are required. Chemical incompatibility with synthesis conditions, the limited selection of readily available phosphoramidites and possible limitations of the stepwise yield after

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incorporation of the modification are narrowing the scope of these methods. However, the construction of long DNA strands functionalized at high density is a difficult task, which cannot be accomplished efficiently by chemical DNA synthesis. In contrast DNA polymerases can synthesize DNA strands several thousand base pairs long and have been utilized in polymerase chain reactions (PCR). Unfortunately, their acceptance of unnatural nucleoside triphosphate analogues carrying the desired functional labels is restricted and frequently not predictable. In general, post-synthetic approaches can overcome these limitations and allow flexible conjugation of different functional molecules to the same modified ODN. Here a small modification is introduced first during ODN synthesis and the labels are then conjugated to the modified ODN by a chemoselective reaction.

The following sections are based on my review "Novel strategies for site-specific covalent labelling of nucleic acids".²⁸

1.2 Methods for labelling DNA

1.2.1 Positioning of labels on DNA

In case of simple applications it is sufficient to label the DNA by linkage at the 5’ or 3’

end. However, more and more applications call for DNA where complicated functional molecules have to be attached site-specifically at positions within the strand, which is more challenging. In principle, chemical modifications can be introduced into ODNs at the nucleobases, the ribose unit or the backbone level.

Many examples have been published, in which the functional molecule replaces one or more nucleotides and therefore is inserted in between the backbone.^29–31 Albeit sufficient for some applications pertubation of helix conformation and incompatibility with most enzymatic reactions are limitations of this approach.

Investigations in the last few years indicate suitable positions for the attachment of modifications within the ribose moiety (Figure 1.1). Modifications at 3’, 4’ and 5’ positions are near the polymerisation reaction sites and often diminish coupling yields.²⁹ 1’

modifications direct into the minor groove and interfere with base pairing and thus have also been rarely used.^{29, 32} Ribose modification at 2’ is synthetically simplest starting with ribose instead of deoxyribose, so this modification is more common for labelling DNA. De- pending on the nature of the linker and the label in most cases the resulting DNA duplex is destabilized. Thereby aromatic labels have an stabilizing effect probably due to groove interactions or π-stacking. These positions allow labelling of ODNs only by chemical synthesis because DNA polymerases, the enzymes that synthesize DNA enzymatically, are

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1.2 Methods for labelling DNA

O OH

HO N

NH O

O R⁵

R^5' R^4'

O OH HO

N N N

NH₂ R⁷

O OH HO

N N N N

NH2

1 5 3

A B C

R^3'

1 3 7 5 9 R^2'

R^1' purine numbering pyrimidine numbering

Figure 1.1: Different imaginable labelling positions for internal labelling. A) Thymidine analogue with 3’, 4’ or 5’ labelling or base labelling (R⁵). B) Adenosine analogue with 1’ or 2’

labelling (R² usually OR). C) 7-Deaza-dA with base labelling (R⁷).

inefficient in processing sugar modified analogues.

The Watson-Crick face of the nucleobase, which is responsible for the interstrand base pairing, should not be touched for modifications. However, the bases have proved to be best suited for labelling purposes, especially for labelled deoxyribonucleotide triphospate (dNTP) analogues, which often are accepted as substrates for DNA polymerases. Modi- fications of the pyrimidines at the 5-position fit well into the major groove and are often used. Modifications at C-8 of purines are not accommodated well into the major groove and disturb at least DNA polymerases by incorporation of such nucleotide analogues.^{26, 33} More elaborated are C-7 modified 7-deazapurine analogues, where the modification points towards the major groove³⁴ which is somehow a prerequisite for efficient processing of modified triphosphates by DNA polymerases.^{26, 35}

1.2.2 Chemical synthesis of functionalized DNA

Since DNA synthesizers evolved to standard equipment, not only the production of rela- tive large quantities of non-modified DNA is feasible but also the generation of chemical modified DNA. Many variations of protecting groups and synthesis conditions allow the incorporation of a broad range of modifications.²⁹ However, most functional groups have to be suitably masked or protected to survive the synthesis conditions throughout the phosphoramidite coupling cycle, which can be an unsolvable challenge.

Most common for internal ODN labelling by phosphoramidites is the 2’ position. The labels are either directly linked to the 2’ oxygen of the nucleotide^{36, 37} (pyrene) or by a carbamate moiety as linker^38–40 (pyrene, dansyl). 2’ amide modified attachment was used for attachment of several aromatic residues.^41–44 In all cases of 2’ labelling the resulting duplexes are thermally destabilized except rarely for aromatic labels which can compensate the steric demand by stacking with the bases.

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O

HO B

A

O OH

O

HO B

N OH

B

Figure 1.2: Labelling of DNA using LNA phosphoramidites. A) Structure of LNA building block to stabilize labelled DNA. B) Structure of LNA building block with directly attached label.

A way to circumvent that 2’ labels disturb duplex formation is the use of locked nucleic acids (LNA, Figure 1.2A). Here the ribose conformation is locked with an additional ring closure from 2’-OH to 4’ by a methylene group as bridge, which leads to increased thermal stability of duplexes and drives the duplex into A-DNA conformation.^{45, 46} LNA monomers placed next to 2-amino labeled nucleotides can thus increase thermal stability of resulting modified oligonucleotide duplexes again.⁴⁷ Moreover, combination of both in one monomer building block by replacing the 2’ oxygen in LNA by nitrogen leads to LNA that can be functionalized at the 2’ amino function and yet gives rise to more stable duplexes (Figure 1.2B).^48–51

The other possibility for labelling ODNs internally is attaching the label to the base.

For the pyrimidine derivatives the 5 position is commonly used, purines can be labelled using either the 7 or 8 position. Depending on the label the conformation and thermal stability of the resulting modified ODN duplex is comparable to the non-modified one.

Some impressive recent examples are the synthesis of glycosylated^{52, 53} ODNs and the development of various dye labelled phosphoramidites.^54–58

Obviously, the scope of modifications is limited by the compatibility of the modifications with the synthesis conditions. Additionally, in certain cases standard phosphoramidites are rapidly oxidized which can be circumvented partially by using H-phosphonates based approaches⁴⁴ which suffer e.g. from longer coupling times.

However, stable modified phosphoramidites enable a one step labelling approach and the ease of subsequent purification are major advantages. Multi-labelling or the generation of long DNA strands is difficult due to limitations of the coupling yield but, nevertheless, has been achieved e.g. for adjacent incorporation of 11 porphyrine labelled phosphoramidites.⁵⁸ Another approach for the introduction of labels harbours the introduction of a functional group during solid phase DNA synthesis by phosphoramidites and subsequent coupling of the label on the solid support. This approach combines the modularity of the post-synthetic labelling by avoiding complicated purification steps with the advantages of organic chemistry on solid support using protected ODNs and standard organic chemistry reaction conditions. Compared to standard amino post-synthetic derivatization, higher

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CPG

O O DMTrO

N NH

O O

I Pd(PPh3)4, CuI, NEt3

DNA synthesis

DMF, 3 h

ODN^PG

R = CH2NHTFA, CH2NHBoc, pyrene R =

N N

O H N

N

N N N

Ru

2+

2 PF₆^- N O

R =

CPG

O O DMTrO

N NH

O R O

DNA synthesis

then deprotection

ODN^PG

R

HN O

NH

O H

N O

S HN NH

O

H H

R = R 13

Figure 1.3: Labelling on solid support. DMTr: 4,4’-dimethoxytrityl, CPG: controlled pore glass, ODN^PG: protected ODN.

yields can be generated with the coupling on solid support due to water-free conditions and protected exocyclic amino groups of the bases.^59–61

A further approach uses the Sonogashira cross coupling reaction between alkyne modified labels and 5-iodo-2’-deoxyuridine (IdU) introduced via solid phase synthesis for the introduction of protected amines, biotin or ruthenium bipyridyl complexes,⁶² pyrene⁶³ or nitroxid spinlabels⁶⁴with excellent coupling yields (Figure 1.3). However, in the latter case low coupling yields for the proceeding DNA synthesis after introduction of the nitroxides were reported. In another case the DNA synthesis has been finished on the solid support before in a combinatorial approach 22 different alkynes (mainly alkyl residues, alcohols, aromatic residues, steroids) have been coupled by the Sonogashira cross coupling reaction to an intrastrand located IdU with good yields.⁶⁵ More recently ethinyl cyanine dyes (Cy3 and Cy5) have been coupled to the 5’ end using the same method.⁶⁶

1.2.3 Enzymatic synthesis of functionalized DNA

One of the advantages of using DNA for construction of artificial structures is that many enzymes modifying DNA are known. Ligases, kinases, and restriction endonucleases can be used for further processing and DNA polymerases for the construction of multi-labelled DNA. By using nucleotide analogues DNA polymerases can incorporate modifications into DNA or even amplify DNA in a polymerase chain reaction (PCR). For the acceptance of modified triphosphates by the DNA polymerase the position of the label is important. As

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NH O

HN N N

H O

HO

NH O H2N

O NH O

HO

OH OH HO O

HO

OH OH

O

HN O HN N O

H O

6

HN O HN

6

HN O HN O N N HN

O HN

6 O

O 6

HN O HN

6 NH₂ H2N HN

O HN

6 O OOC

4 R

NH O O

O 4

O AA S

NH HN O

Vent exo- Taq, Vent, Pfu, rTH Taq, Vent, Pfu, rTH

Taq, Vent, Pfu, rTH

KOD Dash

KOD Dash KOD Dash

KOD Dash

KOD Dash KOD Dash

KOD Dash

R R

6

1 2 3

4

5

6 7

8

9 10

11

Figure 1.4: Overview about successful usage of different labelled triphosphates in PCR. The employed DNA polymerases are indicated. Aside from1all labelled triphosphates were applied without their natural counterpart. R: 5-substituted-dUTP or 7-substituted 7-deaza-dATP. AA: amino acid: Arg, Gln, His, Leu, Lys, Phe, Pro, Ser, O-Bn-Ser, Thr, Trp, Asp, Glu, Cys.

mentioned before modifications at C5 of pyrimidines and C7 of 7-deazapurines are often tolerated, whereas suitable modifications at C8 of purines are only rarely known. Only one example is known, where an imidazolyl moiety at C8 of 2’-deoxyadenosine (dA) was successfully used in PCR.⁶⁷

Incorporation of one or even multiple consecutive nucleotide analogues is possible for a broad range of modifications in primer extension reactions.^{26, 68–72} More challenging is the use of these analogues in PCR, where the nucleotide analogues get incorporated and serve as a template at the same time for the generation of large amounts of modified DNA. Hence, modified nucleotides which can be successful incorporated into DNA during PCR give best insights into the structural elements best accepted by DNA polymerases. Sometimes full replacement of the natural triphosphate is not possible. By mixing natural and labelled triphosphate the PCR though yields the desired length of product, which however is not completely labelled. This approach has been used first for labelling of PCR products with biotin (biotin-dCTP) or digoxigenin (DIG-dUTP)⁷³ as well as for fluorescence labelling by 7-deaza-dA labelled with trans-stilbene (Figure 1.4: 1).³⁵ In the latter example the

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ratio between natural and unnatural triphosphate could be increased up to 3:2 resulting in approximately 3% of the bases getting labelled. But with higher ratios no more PCR product was observed.

More impressive are examples where the nucleotide analogue completely replaces its natural counterpart and allows defined programmed labelling of all bases of a type. The first example with systematic investigations is described by Sakthivel and Barbas.⁷⁴ They had to discover that the acceptance of the nucleotide analogues heavily depends on the structure of the label and the linker (Figure 1.4: examples2-4). Also four different DNA polymerases were tested, whereas rTh DNA polymerase from Thermus thermophilus showed best results. In general, family B DNA polymerases have been found to have a broader substrate spectrum with best candidates such as Vent (exo-), Pwo and KOD Dash DNA polymerases.26, 35, 74, 75 Family A polymerases likeTaq andTTh DNA polymerases appear to be less suited for the incorporation and amplification of modified substrates.

Another systematic study about linker rigidity revealed that analogues with alkyne and E-alkene linker were incorporated by Taq DNA polymerase, while analogues labelled by alkane andZ-alkene linkers were not.⁷⁶ More recently Sawaiet al. investigated the incorporation dependencies mainly of amines conjugated with different linkers to the base.⁷⁵ Five DNA polymerases were used as well as 13 different thymidine analogues and their 2’-deoxycytosine (dC) counterparts. Unfortunately it is difficult to find clear trends but some suggestions can be made. Free amines near the base were not incorporated well; the positive charged amine is only accepted, if a long linker separates it from the base. The acceptance depends also on the nucleotide. For the thymidine analogues short rigid linkers as alkenes and alkynes without positive charge were better accepted than for the dC counterparts, on the other hand 2-oxoethyl linkers were better accepted for the cytosine analogues than for the thymidine counterparts. In general, modifications with a possible strong impact on the active site of the DNA polymerase as positively, negatively charged or bulky groups are best accepted with long flexible linker arms (Figure 1.4: examples 5-8).⁷⁷ Interestingly, modifications attached by thiourea linkage on long linkers are poor substrates for DNA polymerases.⁷⁷ More illustrative examples of one modified dNTP analogue are the successful incorporation and amplification of several different amino acid,⁷⁸ maltose and lactose,⁷⁹ and acridone⁸⁰ labelled thymidine analogues by KOD Dash DNA polymerase in PCR (Figure 1.4: 9-11).

For several applications like the generation of efficient aptamers, DNA catalysts, or new sequencing approaches high density functionalization of every base might be essential.

More than one substitution of natural triphosphates has been achieved several times.^{33, 67, 76}

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Replacement of all natural dNTPs has been published recently by Famulok et al.²⁶ One problem they encountered was unsuccessful melting of the duplex strands during PCR under standard conditions.⁸¹ As they state, this might derive from the use of nucleobase- alkynylated dNTPs which are known to increase the melting temperature of resulting duplex DNA strands. Additionally, modification-induced formation of stable secondary structures was envisioned.^{34, 82} After combinatorial testing of up to five additives the combination of DMSO, formamide, betaine and tetramethylammonium chloride showed the best results for PCR product formation of DNA that constitutes entirely of modified building blocks.⁸¹ Additionally, the melting temperature during the PCR program was also increased to 99^◦C requiring the use of exceptionally thermostablePwoDNA polymerase.²⁶

1.3 Post-synthetic labelling of DNA

1.3.1 Prerequesites for the labelling reaction

Post-synthetic labelling consists of two steps (Scheme 1.1). In the first step a small reactive anchor is introduced during solid phase DNA synthesis or by enzymatic incorporation of nucleotides by DNA polymerases. The advantage of the small anchor is, that it better withstands the synthesis conditions during DNA synthesis and is better accepted by DNA polymerases as unnatural substrate than a complex and potentially large label. In a second step a chemoselective reaction is used to connect the reactive anchor site-specifically with the label. In general, due to the modular preparation post-synthetic approaches allow flexible conjugation of different functional molecules to the same modified ODN.

Albeit, the functionalization of biomolecules is a challenging task since they exhibit diverse reactive functional groups like amines, alcohols, thiols and others. To enable chemoselective labelling the reaction partners of the ligation reaction have to be unreactive towards this entities. On the other hand the ligation reaction has to work smoothly at low temperatures in water. Smoothly means nearly no byproduct formation, preferably high yield and high intrinsic kinetics of the chemical reaction. Sharpless outlined most of this issues and created the term "click chemistry" in 2001.⁸³

Not only for in vivo applications non toxicity of reaction partners or catalysts and the kinetics of the reaction become important, since most ligation reactions follow second- order kinetics, and consequently, their rates depend on the concentrations of the two reactive components and the second-order rate constant.⁸⁴ While the concentrations of the involved species can be controlled to some extent inin vitrosettings,in vivothe target biomolecules are often at low concentrations. Due to the low water-solubility of many labels

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1.3 Post-synthetic labelling of DNA

O O

DMTrO N

NH O

O

N P O CN reactive

group

O OH

4-O₉P₃O N NH O

O reactive

group

DNA polymerase DNA synthesis

reactive group

DNA

complementary reactive group

label

labelled DNA

Scheme 1.1: General concept of post-synthetic DNA labelling

also the concentration of the ligation partner is limited. Biological labelling agents such as monoclonal antibodies typically bind their antigens with biomolecular rate constants that approach the diffusion limit (ca. 10⁹M⁻¹s⁻¹).⁸⁵ Consequently, such reagents can be used at very low concentrations and still bind to their targets at reasonable rates. By contrast, most second-order chemical reactions have rate constants that are 8 - 15 orders of magnitude lower than this.⁸⁴ The reactions discussed in this section have rate constants ranging from 10⁻⁴ (Staudinger ligation)⁸⁶ to 10 M⁻¹s⁻¹ (Cycloadditions).⁸⁷ These rate constants necessitate the use of relatively high concentrations (often high µM to mM) of the labelling reagent when employing bioorthogonal chemical reactions; a parameter which adresses directly the water-solubility of the labelling reagent.

Conventionally for the labelling of DNA amines are used which are coupled with activated carboxylic acids or derivatives thereof. In most cases the amine is usually connected to the DNA. In the labelling reaction it can be distinguished between the exocyclic aromatic amines of the DNA and the alkylamine which builds the reactive anchor. However, the reactivity of the amine depends remarkably on the pH during the labelling reaction, because at neutral pH the amine is protonated and unreactive. At elevated pH values hydrolosis of the activated carboxylic acids increases which leads to suggested pH values between 7.5 and 9.0 for the reaction. Beside amines thiol groups can be used to label DNA. In this case they are reacted withα-haloacetyls,⁸⁸ maleimides⁸⁹ or activated disul- fides.⁹⁰ Also the strong binding to gold can be utilized to label gold nanoparticles with DNA or bind thiolated DNA to gold surfaces.⁹¹ One drawback with thiols is the neces-

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DNA NH₂

DNA SH

DNA O

DNA

DNA N₃ O

O

PPh2

label O label NCS label

O O AG

label O

Cl DNA NH NH S label

DNA NH label O

N label

O

N label O

O S DNA label

O

Br label

O S DNA N

S S

label DNA S S label

N O

O

label O N

O O

label N

O O

label DNA

label N₃ DNA

N N N

label

N H

O DNA

Ph₂P label O O

Staudinger ligation:

Huisgen reaction:

Diels Alder reaction:

DNA

Modified DNA Modified label conjugation product

or or

or DNA

S O

DNA

S N H

N label label H

N NH₂

Table 1.1: Post-synthetic labelling reactions performed on DNA. AG: Activating group.

sary disulfide reduction prior to the bioconjugation reaction. Complicated labels which carry nucleophiles or electrophiles are excluded from the labelling approach using amines or thiols.

An overview of postsynthetic labelling reactions is given in Table 1.1. Recently, 5-(5- formylthiophen-2-yl) substituted dC was incorporated into DNA using the triphosphate by PCR.⁹² Subsequent labelling with hydrazines yielded colored DNA by hydrazone formation using a aniline ammonium acetate buffer (pH 4.5). Although the yields of hydrazone formation in water are only moderate, it is the first example of direct enzymatic incorporation of an aldehyde function into DNA. Below, further reactions are introduced in detail which show a rather high chemoselectivity and bioorthogonality.

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1.3.2 [2+3] Huisgen Cycloaddition

The small azide group combines high intrinsic reactivity with high chemoselectivity. It re- acts for instance with alkynes in a [2 + 3] dipolar cycloaddition and forms stable triazoles reported first by Huisgen.⁹³ Copper catalysis promotes the reaction to proceed at room temperature under aqueous conditions using terminal alkynes resulting in defined regio- chemistry.^{94, 95} Both azides and alkynes are bioorthogonal and therefore soon recognized for bioconjugation and employed extensively.^{96, 97} Albeit for some time the term "click reaction" was used nearly exclusively for the copper catalyzed azide alkyne cycloaddition, nowadays the abbreviation CuAAC is used to differentiate from reactions that also fit in the click chemistry concept.

For DNA conjugation, the first labelling (fluorescein) has been reported for the 5’ end of azido labelled ODNs without copper catalysis.⁹⁸ After 72 h at 80^◦C 91 % labelled ODN could be isolated. Although copper(I) could damage the DNA,⁹⁹ conditions have been found that allow efficient complete conversion within 2 h at room temperature without DNA strand breaks.¹⁰⁰ A terminal alkyne has therefore been sequence specifically attached to the DNA using an aziridine-based cofactor mimic for a methyltransferase reaction and smoothly conjugated with three different azide building blocks.¹⁰⁰ Other examples cover efforts by Carell et al. who developed an octadi(1,7)ynyl 5-substituted-dU analogue for incorporation into DNA and observed complete conversion for up to six adjacent alkynes by labelling with an azido carbohydrate, coumarin azide or fluorescein azide.¹⁰¹ Thereby octadiynyl side chains and stacking triazoles stabilize duplex DNA, whereas single triazole substitutions destabilize the duplex.^{102, 103}

The versatility is shown for the functionalization of ODNs in a modular fashion by using different labels due to protecting groups for the alkyne moiety (Scheme 1.2).¹⁰⁴ A three step labelling has thus been performed using TMS and TIPS protecting groups. Therefore the unprotected alkyne has been labelled first on the solid support after solid phase DNA synthesis. Subsequent deprotection and labelling led to three times labelled ODNs.

Several triphosphates of these nucleotides have been tested in PCR reactions with subsequent CuAAC reaction.¹⁰⁵ Unfortunately, only one nucleotide analogue at the same time could replace its natural counterpart so far. However, the coupling efficiency for the octadiynyl substituted nucleotides was quantitatively for the PCR products as checked by enzymatic digest and HPLC traces.

The CuAAC reaction on DNA has yet been used for the generation of gold wires,^{106, 107} DNA peptide conjugates,¹⁰⁸ DNA circularization and ligation,^109–111 DNA immobilization on glass slides¹¹² and can be accelerated by microwaves.^{109, 113} Recently a protocol for

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O

5'-(GCGC)'-O O-G'TT-O N

N NHBz

O

O N HN

O

O-A'TT-O O

N N

NHBz

O

TIPS

O-(CGC)'-3' TMS

4 4

1. Click:

CuBr, TBTA, sodium ascorbate, benzyl azide, then deprotection of base protecting groups, TMS and cleavage from resin by conc. NH3

N

N N TIPS

N N N N

N 2. Click:

CuBr, TBTA, azido sugar, then deprotection of TIPS with TBAF

N N N N

N

N N N 3. Click: CuBr,

TBTA, azido biotin biotin-NH

DNA

DNA DNA CPG

O OH OH

HO HO

N O

OH OH

HO HO

N

4

Scheme 1.2: 3 ×CuAAC conjugation on DNA. TMS: trimethylsilyl, TIPS: triisopropylsilyl, CPG:

controlled pore glass, TBTA: tris-(benzyltriazolyl-methyl)amine, A’, C’, G’: base protected dA, dC, dG.¹⁰⁴

detecting DNA synthesis in vivo by click chemistry has been published.¹¹⁴ Albeit only available for short time, the click reaction for DNA conjugation has been much investigated and phosphoramidites and triphosphates have been developed for the introduction of alkynes into DNA. The alkyne and triazol moieties do not disturb the DNA duplexes much or even stabilize them and, in principle, complete conversion is achieved for the conjugation within hours at room temperature. The only drawback is the need for the copper ligand catalyst system, which makes the reaction more complicated than the other post-synthetic methods. Additionally copper may interfere in subsequent biotransformations that might be required for further DNA-based construction. CuAAC reactions seem to be unsuited for attaching metal ion chelating labels and are of limited use for inin vivo applications.

Although not yet used for labelling of DNA, efforts of Bertozzi cover the development of a mild copper free azide alkyne cycloaddition. Mild reaction conditions are obtained by using cyclooctyne derivatives in which ring strain release promotes the reaction at low temperatures. Very recently, biarylazacyclooctynone (BARAC) was introduced reaching a rate constant of 1 M⁻¹s⁻¹ for the conjugation.¹¹⁵

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1.3.3 Diels Alder reaction

The Diels-Alder [4 + 2] (DA) cycloaddition¹¹⁶between a conjugated diene and a dienophile is an extremly useful carbon-carbon bond-forming reaction in organic chemistry (see Scheme 1.3).¹¹⁷ DA reactions can be performed under mild conditions without the use of a catalyst, but can be accelerated using Lewis acids. The specific reactivity between both functionalities, no by-product formation and the acceleration of the reaction in aqueous solvents¹¹⁸ make it suitable for covalent bioconjugation.¹¹⁹ In DA reactions with normal electron demand the dienophile has to bear electron withdrawing groups whereas the diene has to be electron rich to facilitate product formation under mild conditions. The DA cycloaddition introduces a stereogenic centre to the conjugation product, however, the stereoselectivity of the reaction is enhanced towards the kinetically favored endo product in aqueous systems.^{120, 121}

Maleimides are one of the most reactive water stable dienophiles with the advantage that numerous labels are commercially available, thus they are in every reported case used as electron poor dienophiles. The high reactivity results from the electron poor double bond and the ring strain which is released during the reaction course. However, maleimides also react smoothly with thiols and they can react with other nucleophiles in the long term, which is problematic for biomolecule labelling.¹²² Hydrolysis of the imide to a open maleamic acid is also reported for pH > 7.5.¹²²

Due to the stability of the diene to biofunctionalities the diene is incorporated into DNA which renders the otherwise insoluble 1,3-dienes water-soluble. Several electron-rich dienes have been employed including anthracene, hexadiene, cyclohexadiene and furan. In 1997, the first examples of DA reaction on nucleic acids showed the principal usability for bioconjugation with RNA resulting in a RNAzyme catalysing the DA reaction between 5’

attached hexadiene or anthracene moieties.^{123, 124}

For post-synthetic 5’ end labelling of DNA cyclohexadiene and acyclic hexadiene phos-

O N

O

O R'

O O

O N O H H

R' N

O O

R' H

H

endo exo

R

R R

+ +

Scheme 1.3:DA cycloaddition occurs under mild conditions between an electron rich diene,i.e.

furan, (cyclo)hexadiene, anthracene, pentadiene, and an electron deficient alkene (for labelling in most cases N-labelled maleimide). In water the reactivity is enhanced and the endo product favoured. R, R’: residues to conjugate.

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P O O O O 3'-ODN

N O

O R

P O O O O 3'-ODN

N O O

R R =

HN O

HN

4 O S

HN NH O

H H

O O NMe2

O OMe

n

n = 112, 448 N

O

O fluorescein

Me

Scheme 1.4: 5’ end labelling of DNA using hexadiene attached to the ODN by phosphoramidite chemistry and various maleimide functionalized labels for DA conjugation.¹²⁵

phoramidites have been prepared and attached to the DNA using standard synthesizer chemistry (Scheme 1.4).¹²⁵ Subsequent labelling with different maleimide functionalized labels and optimized reaction conditions as pH, temperature and label concentrations led to near quantitative conjugation with reaction times ranging from 30 min to 20 h for more complex structures as dyes. Thereby both employed dienes showed compareable conjugation efficiencies. Also surface immobilization could be achieved by the same group.^{126, 127} Addition of copper(II) nitrate allowed for shorter reaction times less than 1 h by using a furan moiety at the 5’ end and conjugation of a benzotriazole dye maleimide for attomole detection using surface enhanced resonance Raman scattering (SERRS).^{128, 129} As a further example 7-vinyl-7-deaza-2-deoxyguanosine 3-phosphoramidite was incorporated into oligonucleotides and subsequent conjugation with different maleimide functionalized labels as carboxylic acid, activated ester, benzophenone, pyrene, TEMPO and biotin was investigated (Scheme 1.5).¹³⁰ Thereby the vinyl double bond and the one in the 5-membered ring of 7-deazaguanine form an electron rich diene, so all labels were attached within 1 h at 0^◦C in aqueous solution quantitatively with a subsequent [1,3] H-shift restoring the deazaguanine. These mild conditions are unrivalled, but not further investigated or employed for DNA labelling. Recently, ODN peptide conjugates were prepared using hexadiene 5’

modified oligonucleotides and maleinimide modified peptides.¹³¹

Few examples report successful DA reaction for internal strand labelling. A furan conjugated to the 5 position of 2’-deoxyuridine (dU) was reported to act as the diene for internal ODN labelling (Figure 1.5A).¹³² After incorporation into an ODN using phosphoramidite chemistry five commercial available fluorescence dyes were conjugated near quantitatively within 3 h at 40^◦C or 4 h at room temperature (r.t.). Several other nucleotide analogues

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O O O

NH N N

O

NH2

P

ODN O

O O 5'-T

N O

O R

O O

O NH

N N

O

NH2

P

ODN O

O O 5'-T

N O

O

R = Me COOH O

O N

O

O O

NO H

N O

HN

O O

S HN NH

O

H H

Scheme 1.5:Labelling of DNA using 7-vinyl-hexadiene attached to the ODN by phosphoramidite chemistry and various maleimide functionalized labels for DA conjugation.¹²⁵

using a furan moiety as diene for the incorporation into ODNs have been developed, but not investigated regarding DA conjugation efficiency on DNA.¹³³ Häner reported DA conjugation using a hexadiene non nucleotide building block reporting lengthy reaction times of 7 d.^134–136 Very recently, a nucleoside triphosphate with base attached hexadiene moiety was employed for enzymatic generation of DNA and subsequent DA conjugation of fluorescent dyes (Cy5 and fluorescein) and oligopeptides with up to 80 % conversion after reaction at 37^◦C for 16 h (Scheme 1.5B).¹³⁷

O O

O N

NH O

O O

NH O

ODN ODN

O O

O N

NH O

O O

ODN ODN

A B O

Figure 1.5: Labelling of DNA using internally diene modified ODNs. A) Incorporated by solid phase DNA synthesis. B) Incorporated by DNA polymerases.

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1.3.4 Staudinger ligation

The Staudinger ligation is a bioorthogonal conjugation reaction which needs no catalyst to proceed. Invented by Bertozzi in 2000¹³⁸the reaction is based on the Staudinger reduction, wherein an azide is reduced by a phosphinevia an iminophosphorane intermediate.¹³⁹ In the ligation reaction an intramolecular acetylation of the iminophosphorane takes place before competing hydrolysis occurs, thus conjugating the phosphine with the azide by forming a stable amide bond (Scheme 1.6).

While the azide is small in size, stable under a wide variety of conditions biomolecules can tolerate and has a high intrinsic reactivity, but is unreactive to nearly all functional groups of biomolecules, the phosphine suffers from its low water solubility, bigger size and susceptibility to oxidation. Nevertheless the ligation reaction is selective and the phosphine moiety bioorthogonal to almost all biofunctionalities and thus has been shown to work selectively in vivo right from the first publication for labelling cell surface azido glycoconjugates with biotin.¹³⁸ Later examples show the general applicability to label biomolecules like peptides, proteins, carbohydrates and DNA.^140–146

Mechanism and kinetic of the Staudinger ligation have been thoroughly investigated by Bertozzi et al. in 2005.⁸⁶ A detailed proposed reaction mechanism is depicted in Scheme 1.7. They presume that the early steps k1 and k2 of the reaction mechanism are similar to the classical Staudinger reduction which can occur as a side reaction depending on the substrates used in the reaction (Stepk₆). Typically iminophosphorane14undergoes a fast intramolecular reaction (Step k₃) to proposed intermediate 15 which could not be detected yet, since in presence of water hydrolysis occurs very fast yielding the final ligation product 17. In absence of water compound 19 can be isolated which indicates likely existence of 15. Reaction of isolated compound19 with water again yields ligation

HN O R

OMe O

PPh2

HN O R

OMe O

P Ph Ph

N R'

HN O

R P

N O

Ph Ph

R' H

N O R

NH O

P Ph Ph

R' O N3 R'

N2

-OMe

OH^-

Scheme 1.6: Simplified reaction mechanism of the Staudinger ligation. R and R’: residues to ligate.

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N3 R +

N₂

H2N R Staudinger

reduction

ligation product anhydrous

conditions

H2O LGO^-

LGOH

+

amine side product H₂O

P N O HO

Y

R P

Y P

Y

O OLG

N N N R P

Y

O OLG N R

P N O

Y

R P N

O LGO

Y

R O

OLG

P

Y

O OLG

P

Y

O O

O N R H Staudinger

ligation

= Bn, Ph, p-PhOMe or p-PhNO2

= Me, Et, ⁱPr, tert-Bu, p-PhOMe, p-PhMe, Ph, p-PhF, p-PhCl or p-PhNO2

= NMe2, OMe, OH, Me, Br or NO2

R LG

Y

k1

k₆

k₄ k3

k2

k5

5

12

13 14

15

16 17

18

19

Scheme 1.7:Detailed reaction mechanism of the Staudinger ligation.⁸⁶ Unless otherwise stated R = Bn, LG = Me, Y = H.

product17, presumably across the formation of intermediate15.

For the kinetics they used a basic system consisting of benzyl azide and phosphine 12 (R = Me, Y = H, Scheme 1.7) in acetonitrile and 5 % water. It turned out that the rate limiting step is the initial reaction wich is most likely irreversible and the reaction is bimolecular with a second order rate constant of 2.5 · 10^-3M⁻¹s⁻¹. They pointed out some solvent effects whereas more polar solvents like DMSO or protic solvents like methanol increased the reaction rate. The reaction rate in aprotic solvents can be further increased with a higher water content. With methanol and DMSO the rate constant reached 3.6 · 10^-3M⁻¹s⁻¹.

The variation of the substrate structure elucidated some effects on the reaction rate.

While the ester leaving group (LG) has nearly no effect on the reaction rate (except of tert.-butyl ester which favours Staudinger reduction side reaction), substitutions to the phenyl rings change the electronic situation on the central phosphor atom and thus effect

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ODN(18mer)

H₂N H

N ODN(18mer) O

N3

6 ON

N₃

O O O

O O

HO

HN O

NH O

O OMe PPh₂

HN ODN(18mer) O

HN O 6 PPh₂ O O

NH O HN COOH

O

HO O

COOH

Scheme 1.8: 5’ terminal labelling of ODNs by Staudinger ligation. Reaction conditions for Staudinger ligation: Na2CO3/NaHCO3 buffer (pH 9.0), DMF.¹⁴⁶

the reaction rate. Electron withdrawing groups decrease the reaction rate (Y = NO2, Br) whereas electron donating groups accelerate the reaction (Y = NMe2, OMe, OH).

Interestingly, when aryl azides instead of benzyl azide were used, a stable intermediate was quantitatively formed within 5 min which has been adressed to intermediate 14 and only slowly hydrolises (8 to 48 h). In this case the aryl moiety might stabilize the negative charge on nitrogen in intermediate 14.

For labelling DNA by the Staudinger ligation either the azide or the triphenylphosphine moiety has to be incorporated into DNA which is a challenging task. Both functional groups are instable to normal phosphoramidite DNA synthesizer conditions. However, two examples of Staudinger ligation on DNA are known. First, 5’ terminal labelling with fluorescein is reported, the fluoresccein being attached to the triphenylphosphine moiety using a small linker (Scheme 1.8).¹⁴⁶ The azide was coupled post-synthetically to the DNA using a azido active ester which was connected to a terminal amino group of the employed ODN. For the Staudinger ligation 90 % yield was reported after incubation for 12 h at r.t.

In the second example a enzymatic approach to attach the azido function to the DNA is used(Scheme 1.9). Therefore, an azido labelled substrate for a DNA methyltransferase was used and sequence specifically attached toN-6 of dA in dsDNA. By subsequent Staudinger ligation with different substrates biotin could be conjugated within 14 h at 40^◦C¹⁴⁷ or phenanthroline for subsequent copper(I) induced strand scission.¹⁴⁵

In the classical Staudinger ligation the bulky triphenyl phosphine remains in the ligation product which might cause problems as steric hindrance, disturbing natural biomolecular

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O OH N

N N N N

NH2

N₃ H

O O O

N N N N

HN

5'-DNA

DNA O

3'-DNA T O DNA O

O O

N N N N

NH2

5'-DNA

DNA O

DNA3'- T O DNA

O OH

N N N N

NH2

N₃ HN

PPh₂ O

OMe O

HN H O

RN _n

O O O

N N N N

HN

5'-DNA

DNA O

3'-DNA T O DNA

O OH

N N N N

NH2

H N N

OMe PPh2

O O

NH O

NH R

R = biotin or phenanthroline n = 2 or 3 7

Scheme 1.9:Internal labelling of ODNs by Staudinger ligation. Reaction conditions for Staudinger ligation: NaOH, DMF.^{145, 147}

structure or preventing multi labelling in close proximity. A possible solution is the traceless Staudinger ligation invented in parallel by Bertozzi and Raines in 2001.^{148, 149} Here the (thio)ester functionality is reversed: the leaving group consists of the phosphine and is cleaved, whereas the carboxy part is transfered and forms a native amide bond at the ligation reaction site (Scheme 1.10). Thus the traceless Staudinger ligation is especially useful to ligate oligopeptides to form larger polypeptides or peptide conjugates,^150–152but has been also used for the labelling of biomolecules which is in particular useful since a

X O R

PPh2

X O R

P NH PhPh

P N PhPh R'

R' R

O X

P Ph Ph

XH O N

H

R' R

O R' N₃

+

+ N2

H2O

Scheme 1.10:Simplified reaction mechanism of the traceless Staudinger ligation. R and R’:

residues to ligate, X = O¹⁴⁸ or S.¹⁴⁹

Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction