Contents
Scheme 2.3. Click chemistry to generate fluorescent nucleoside analogs from non-fluorescent precursors
There are several examples of click chemistry mediated incorporation of fluorophores into DNA. The two basic methods utilized for this purpose are pre-synthetic and post-synthetic modifications of DNA. In this respect, we will discuss some recent examples of modifications at the DNA bases achieved via these two methods below.
2.4.1. Pre-Synthetic Modification of DNA
The term “pre-synthetic modification” denotes the desired modification on a nucleoside prior to the incorporation into DNA. In the field of the click reaction mediated synthesis of base modified fluorescent nucleosides/nucleotides, major contributions are done by Seela and co-workers. In 2012, they have reported two pyrrolo-dC (dC = deoxycytidine) click adducts (2.21, 2.22, Figure 2.5) and incorporated into oligonucleotides for DNA mismatch detection.55 According to the authors, the oligonucleotide probes containing these compounds were able to discriminate between matched and mismatched DNA base pairs.
Figure 2.5. Fluorescent nucleoside click adducts reported by Seela and co-workers.
Seela and co-workers also reported several pyrene-labeled nucleosides (2.23-2.27, Figure 2.5) and incorporated into oligonucleotides.56, 57 They have reported that the nucleoside 2.23 containing a short linker is destabilizing to the DNA duplex formation, while the nucleosides 2.24 and 2.25 with long linkers improve the DNA stability.56Also, the dC click conjugates (2.24, 2.26) exhibited superior fluorescence emission properties than the 7-deazaguanosine derivatives (2.23, 2.25, 2.27), both in single-stranded and double-stranded DNA.57
2.4.2. Post-Synthetic Modification of DNA
Incorporation of fluorophores via pre-synthetic modification have certain limitations. For example, many fluorophores susceptible to decompositions under basic conditions and at deprotection step used during the solid-phase oligonucleotide synthesis or during the PCR process. Moreover, incorporation of bulky adducts into DNA can be difficult due to the steric hindrance. In these contexts, the post-synthetic modification is most probably the ideal method for DNA modification. The term “post-synthetic modification” denotes that the desired modification has been done on the DNA strand within a complex biological medium.43, 58 In this respect, we will discuss a few examples in this section below.
In 2006, Carell et al. reported multiple post-synthetic labeling of alkyne-modified DNA.59 They synthesized two modified uridine nucleosides (2.28, 2.29, Figure 2.6) and incorporated into ODNs for the generation of ODNs bearing alkyne reporter groups in various densities.
Afterward, they performed click reaction between the alkyne bearing duplexes of these ODNs and various azides (2.19, 2.30, 2.31, Figure 2.6). A low conversion to products was reported for the ODNs containing the nucleoside with short alkyne linker (2.28), while complete high- density conversion was reported for ODNs containing the nucleoside with long flexible alkyne linker (2.29).
Figure 2.6. Alkyne bearing nucleosides and organic azides utilized by Carell et al. for post- modification of DNA.
In 2010, Seela et al. employed fluorogenic azides, 9-azidomethyl anthracene (2.34) and 3- azido-7-hydroxycoumarin (2.19) for post modification of ODNs containing octa-(1,7)-diynyl- 8-aza-7-deaza-2′-deoxyadenosine (2.32, Figure 2.7).44 According to the authors, the modified ODNs showed enhanced fluorescence emission as well as stability upon fully matched duplex formation. They have also compared the fluorescence emission properties of click compounds obtained by reaction between the free alkyne group of 8-aza-7deaza-2′-deoxyadenosine (2.32)
and 7-deaza-2′-deoxyadenosine (2.33) with both of the fluorogenic azides (2.19, 2.34). It is interesting that the 8-aza-7-deaza-2′-deoxyadenosine click compounds did not exhibit fluorescence quenching in their analysis, while the 7-deaza-2′-deoxyadenosine click compounds exhibited quenched emission most possibly due to the charge transfer between the nucleobase and the fluorophore.
Figure 2.7. Alkyne bearing nucleosides, 8-aza-7deaza-2′-deoxyadenosine, 7-deaza-2′- deoxyadenosine and the fluorogenic azides utilized by Seela et al. for post-modification of DNA.
In 2010, Wagenknecht et al. reported successful incorporation of Nile Red dye into DNA post-synthetically (Figure 2.8).60 For that purpose, they have utilized the in-situ generation of azide in the DNA by reaction with a nucleotide containing 5-iodo-2′-deoxyuridine with sodium azide, which was followed by a click with an ethynyl modified Nile Red (2.35) to yield the modified DNA.
Figure 2.8. Schematic representation of post-synthetic incorporation of Nile Red dye into DNA reported by Wagenknecht et al.
The use of bioorthogonal chemical reporters in order to monitor biomolecules in living systems has drawn much attention among researchers lately.61-65 Because of the unobtrusive nature of click substrates (azides and alkynes), click chemistry is a powerful candidate for generation of such reporters without perturbing the targets biological functions. However, performing Cu(I) catalyzed click reactions within live cells is not a safe practice even successful examples have been demonstrated.66, 67 Copper ion impurities from the post- synthetic or in vivo modification can become a cytotoxic threat even when present in trace amounts. Hence, the development of copper-free “click” type reactions and alternatives for post-synthetic modification of nucleic acids is a current research topic in demand. Such types of reactions include strain-promoted54, 68, 69 and other 1,3-dipolar cycloadditions,70, 71 Diels- Alder reactions with normal72 and inverse electron demand,73, 74 reductive aminations,75 thiol- ene additions76, 77,and Suzuki-Miyaura-type coupling reactions.78, 79
2.5. Background
Polarity sensitive fluorescent molecules are ubiquitous for sensing of biomolecules and studying inter-biomolecular interactions inside a cell.80-82 In particular sensing of the local microenvironment of DNA is highly important in connection with the detection of DNA mutations causing a deleterious effect on cellular survival, high throughput screening, and many other biotechnological applications.83-85 All these events in DNA rely on novel fluorescent probe either as bare or unnatural fluorescent nucleosides or fluorescently labeled natural nucleosides.1, 86-88 Though many such probe systems in relation to DNA have been reported but the probes suffer from fluorescence quenching by neighboring nucleobases or short wavelength emission or poor microenvironment sensitivity.1, 86 Therefore, designing of novel emissive probes, particularly, fluorescent nucleosides with unique fluorescence properties, extreme sensitivity to change in DNA microenvironment and interactions are highly desirable. Among the three approaches, linking a fluorophore in nucleoside bases is the major approach to generate fluorescent nucleoside useable for DNA sensing.1, 2, 83-91 As a result of tremendous research efforts, a large number of fluorescently labeled nucleosides and corresponding oligonucleotide probes have been designed and utilized to a variety of applications that include probing DNA hybridization,92, 93 typing single nucleotide polymorphism (SNP),7, 94, 95 and monitoring the interbiomolecular interaction,96-98 to name a few. However, the majority of the reported environmentally sensitive fluorescent nucleosides
exhibited single band emission that senses the differences in micropolarity either by a change in emission intensity or wavelength.17, 90, 99-102 Among these, Saito’s ESF nucleosides100, 103,
104(1.224-1.228, Figure 1.27, Chapter 1)are highly attractive for monitoring the micropolarity changes within DNA. However, often the majority of such probes suffer from several shortcomings such as poor microenvironment sensitivity and low quantum yields.1, 83-88, 100, 103- 105
Therefore, to overcome these limitations, the concept of two-band emission would be more advantageous over commonly utilized single-band fluorescent probes/nucleosides.106-108 Thus, recording a ratio of the intensities at two wavelengths would allow ratiometric sensing which is more advantageous than sensing based on single wavelength emission.109, 110 Basically, ratiometric sensing results in an intrinsically calibrated emission response.106-108 Ratiometric probing of DNA, though reported, but is based on labeling of DNA by two interacting dyes such as FRET pair or excimer/exciplex pair.93, 95, 111, 112 However, labeling with two dyes is difficult, time-consuming as well as highly uneconomical.93, 95, 106-110 On the contrary, a single fluorophore with dual emission property would be much more beneficial.113-115 Highly increased dipole moment and dipole-dipole interactions in the excited state allow such fluorophores to be able to sense the changes in local micropolarity within a biomolecular microenvironment or in the cell.80-82 Therefore, dual emissive fluorophores are very useful as a ratiometric probe because they offer facile and straightforward quantification of a biomolecular event through the ratio of their two bands. However, due to the scarcity of such fluorophores that display dual emissions and the difficulties in their syntheses, the phenomena of dual emission based sensing of biomolecular events are poorly explored, especially, in the field of DNA analysis.116-119 With a poor literature reports and the unique ability for sensing the change in the microenvironment of DNA biomolecules, the design of dual emissive modified nucleosides that can control the equilibrium between two excited states at ambient temperature without changing the solvent properties is an unavoidable research area.
2.6. Aim and Objective
As a part of our continuous research efforts in the design of solvofluorochromic molecules/biomolecular building blocks, 120-124 we thought that it would be worthwhile to design dual emissive modified nucleosides. Based on our experience, literature reports and wider applicability we considered the design of C5 substituted uridines as model nucleoside probes useable for DNA analysis in the future.45, 89, 120, 125-134 However, there is no report wherein C5 position of 2′-deoxyuridine is linked by an electron donor unit as a post-
synthetically modifiable functional group which effectively can generate a modulated fluorescence property of a fluorophore if attached at the terminus or the terminal alkyne can be reacted with a fluorophoric azide functionality. Previously, we have shown the
“installation/modulation of fluorescence response” of various small fluorescent molecules and an interesting dual emission behavior from pyrene when attached to N,N-dimethylanilino triazole donor unit.135 Inspired by our previous result and motivated by the importance of dual emitting probe for DNA analysis,116-119 we thought that it would be worthwhile to generate a set of fluorescent 2'-deoxyuridine nucleosides which could show interesting intramolecular charge transfer property or dual emission. We further thought that attaching an electron donor phenylacetylene unit as a post-synthetically modifiable functional group at the C5-position of 2'-deoxyuridine would be beneficial to generate a set of fluorescent 2'-deoxyuridines with modulated fluorescence property of a fluorophore via azide-alkyne cycloaddition reaction.45,
121-124, 129-134 Furthermore, the same nucleoside, if incorporated into DNA, can offer the opportunity of generating fluorescent oligonucleotide probes via post-synthetic click reaction with modulated fluorescence property. Following the aforementioned design logics the research was aimed as below:
(a) The design and synthesis of 5-(3-((4-ethynylphenyl)(methyl)amino)propynyl)-2'- deoxyuridine a possible post-synthetically modifiable nucleoside.
(b) Its application to generate a set of triazolyl fluorescent 2'-deoxyuridines generated via Sonogashira cross-coupling reaction and azide-alkyne cycloaddition reaction.
(c) Study of photophysical properties of a few such fluorescent 2'-deoxyuridines revealed interesting solvatochromic photophysical properties corroborating our design concept.
(d) Theoretical study to support the experimental photophysical properties of three fluorescent uridines by TDDFT calculation.
(e)