1.4. Artificial Base-Pairs Based on Hydrogen Bonding Interaction
1.4.1. Purine Base Analogues
2-Aminopurine (2-AP) as Adenine Base Analogue: 2-aminopurine (2-AP) (1.23), a structural isomer of adenine which is highly fluorescent was first demonstrated by Stryer and colleagues.15 As it is structurally similar to adenine (6- aminopurine), 2-AP is a non-perturbing substitution of adenine and also forms thermodynamically stable base pairs with thymine and uracil through hydrogen bonds in DNA and RNA helices respectively (Figure 1.5). 2-AP is also forms a base pair
with cytosine, in contrast to adenine, which is the basis for 2-AP’s mutagenicity. 2-AP can be incorporated into both DNA and RNA oligonucleotide sequences in a site- specific manner. The quantum yield of 2-AP is highly sensitive to its microenvironment and insensitive to base pairing and other H-bonding interactions.
Hence, it serves as an efficient reporter nucleoside analogue in an oligonucleotide probe to detect subtle conformational changes in nucleic acids.
N N H N
H
NH H
2-aminopurine (2-AP) 2-AP/U pair 2-AP/C mispair
1.23 1.23a 1.23b
O
OH HO
O OH
OH N N
O O
N N H N
H
NH H O
HO
HO H
O OH
OH N N
O HN
H N
N H N
H
NH H O
HO HO a. b.
Figure 1.5: (a) Structure of 2-Aminopurine and its (b) Hydrogen bonding pattern with uracil and cytosine.
Other base analogues of adenine include 2-aminoadenine (2,6-diaminopurine) and 7-aminopropargyl-7-deaza-2,6-diaminopurine. 2-aminoadenine was synthesized and introduced into oligonucleotides by Chollet et al.,16 which pairs up with thymine through additional hydrogen bonds and also introduces subtle changes in the minor groove of DNA. Oligonucleotide hybridization probes containing 2-aminoadenine have shown increased selectivity and hybridization strength during DNA-DNA hybridization to phage or genomic target DNA. 2-Aminoadenine has been used to probe minor groove detection during the treatment of DNA by 12 restriction endonucleases wherein inhibition of cleavage is the result for several restriction enzymes. 7-aminopropargyl-7-deaza-2,6-diaminopurine has been synthesized and incorporated into oligonucleotides by Brown et al., and its pairing property has been studied. When paired up opposite to thymine, it was found to have similar thermodynamic stability as that of C:G pair.17
ATP/AMP Analogues: Other fluorescent analogues of adenine have been
developed and used primarily as probes of AMP/ATP binding by enzymes (Figure 1.6). These other base analogues include etheno-ATP18a-c and lin-benzo-AMP18d, which are analogues of ATP and AMP, respectively. Another ATP analogue,
formycin 5'-triphosphate18e, has been used as a substrate analogue for adenylate cyclase. These analogues have been used primarily in studies of nucleotide cofactor binding to enzymes.
N
N N
N N
H N
H
N N
H
H NH2
H
N N
N N H H N H
Etheno adenosine Lin-benzo-adenosine Formycin 1.24 1.25 1.26 O
OH HO
O OH
HO O
OH HO
Figure 1.6: Structures of adenine base analogues used to construct fluorescent nucleoside and nucleotide analogues- Etheno-adenosine, lin-benzo-adenosine and formycin.
Pteridine Analogues of Adenine and Guanine: Pteridine is a class of heterocyclic
compounds composed of fused pyrimidine and pyrazine rings.19 Pteridine analogues of adenine and guanine have also been synthesized. Reported pteridine adenine analogues include 4-amino-6-methyl-8-(2-deoxy-β-D-ribofuranosyl)-7(8H)-pteridone (6-MAP) (1.27, Figure 1.7a) and 4-amino-2,6-dimethyl-8-(2’-deoxy-β-D- ribofuranosyl)-7(8H)-pteridone (6-DMAP) 20a (1.28, Figure 1.7a). They have been synthesized and incorporated into short stretches of oligonucleotides in order to study their pairing and fluorescence properties. The pteridine-containing oligonucleotides have melting temperatures similar to that of the unmodified control oligonucleotides thus showing that they induce minimal changes in DNA when incorporated as an adenine analogue. Two pteridine guanine analogues, 3- methyl isoxanthopteridine (3- MI) (1.29, Figure 1.7b) and 6- methyl isoxanthopteridine(6-MI) 20b,c (1.30, Figure 1.7b) have been synthesized and incorporated into DNA. After incorporation into DNA, both the pteridine adenine and guanine analogues display significant quenching of fluorescence intensity, increased complexity of fluorescence decay curve and decreased mean fluorescence lifetime. The degree of quenching of fluorescence intensity of pteridine adenine and guanine analogues correlate with the number and proximity of purines in the oligonucleotide. The degree of quenching did not increase
upon formation of double stranded oligonucleotides but the complexity of decay curves increased and mean fluorescence lifetimes decreased.
N N
N N O
O CH3 NH2
N N
N NH O
O
NH2 H3C
3- methyl isoxanthopteridine (3-MI) 20b 6- methyl isoxanthopteridine (6-MI)20c N
N N
N O
NH2
CH3 H3C
4-amino-6-methyl-pteridone (6-MAP) 20a 4-amino-2,6-dimethyl-pteridone (6-DMAP) 20a N
N N
N O
NH2 H3C
a. Pteridine analogs of adenine
b. Pteridine analogs of guanine
1.27 1.28
1.29 1.30
O OH HO
O OH HO
O OH
HO O
OH HO
Figure 1.7: Structure of Pteridine analogues of (a) adenine and (b) guanine 1.4.2. Pyrimidine Base Analogues
Benzo[g]quinazoline Based Thymine Base Analog: Godde and colleagues have synthesized bases with extended aromatic domains that increase third strand binding through stacking interactions.21 One of the polycyclic aromatic base analogues of thymine, benzo[g]quinazoline-2,4-(1H,3H)-dione(1.31, Figure 1.8), is found to display strong fluorescence emission centered at 434 nm (ΦF ~ 0.82) and two major excitation maxima (260 and 360 nm).21 Formation of the triple helical structure using a third oligopyrimidine Hoogsteen strand that contain this fluorescent thymine analogue, results in a shift of the fluorescence emission maximum to shorter wavelengths and a decrease in fluorescence intensity.21 In a duplex, it does not produce any significant changes in fluorescence properties.21 Thus, the sensitivity of this base analogue to the helical conformation allows selective detection of triplex over duplex formation.
Thymine Analogue: Another thymine analogue 5-methyl-2-pyrimidinone (1.33, Figure 1.8) has been synthesized and used in early studies of DNA duplexes. This base analogue does not pair well with adenine, however, using time-resolved fluorescence decay measurements it has been shown that the predominant state of the base in the context of a DNA oligonucleotide is stacked so that its fluorescence is efficiently quenched.22
O OH HO
1.31 1.32 1.33 Benzo[g]- and benzo[f]-quinazoline-2,4-(1H,3H)-dione
N HN
O
O N
NH O
O N
N H
O H3C
5-methyl-2-pyrimidinone Thymine Analogues
O OH HO
O OH HO
Figure 1.8: benzo[g]/[f]quinazoline-2,4-(1H,3H)-dione and 5-methyl-2-pyrimidinone as thymine analogue.
Benzo[g]quinazoline Based Cytosine Base Analogue: A 2'-O-Me ribonucleoside
derivative of 4-amino-1Hbenzo[g]quinazoline-2-one (1.34, Figure 1.9) has also been synthesized based on the same heterocyclic benzo[g]quinazoline (1.35, Figure 1.9) design and used as a novel fluorescent cytosine base analogue probe.23 This cytosine base analogue exhibits a fluorescence emission centered at 456 nm, characterized by four major excitation maxima (250, 300, 320 and 370 nm) and a fluorescence quantum yield of ΦF = 0.62 at pH = 7.1. The fluorescence emission of this probe shifts from 456 to 492 nm when pH is decreased from 7.1 to 2.1. The pKa (4.0) of the probe is close to that of cytosine (4.17). This probe has been used to detect the protonation state of base triplets in triple stranded structures.
O HO
HO OCH3
N
HN O
O
O HO
HO OCH3
N
N NH2
O
1.34 1.35
Y N
N N
O N
N N O N
N H
H H H
O N
N N
O H N O
O N
N N
O H O NH3
G
3,5-diaza-4-oxophenothiazine (tC) (X = S) and 3,5-diaza-4-phenoxazine (tCO) (X = O) tC/tCO
N N
O NH H3C
Pyrrolo-dC
1.36 1.37 1.38 1.39
O HO
HO O OH
OH O
OH HO
O OH
HO O
OH HO
A C D E
B
Figure 1.9: Structures of 1-(2-O-methyl-β-D-ribofuranosyl)- benzo[g]quinazoline and 1-(2-O-methyl-β-D-ribofuranosyl)-4-amino-1H benzo[g]quinazoline-2-one.
Tricyclic Cytosine (tC): Norden and colleagues have described a new cytosine base analogue, 3,5-diaza-4-oxophenothiazine or tricyclic cytosine (tC) (1.36), which can form a specific base pair with guanine (Figure 1.10). Like the benzo[g]quinazoline base analogues, this base maintains its relatively high quantum yield (ΦF = 0.20) even after incorporation into single and double stranded oligonucleotides, like artificial peptide nucleic acid (PNA) biopolymers and RNA- DNA duplexes.24
Elaboration of the tC(O) scaffold can yield a nitroxide spin labeled compound (1.31) that may be used for EPR measurements25a and the “G-clamp” which has increased binding affinity to guanine (A, C, D)25b-e (Figure 1.10).
Figure 1.10: 3,5-diaza-4-oxophenothiazine (tC) (X = S) and 3,5-diaza-4-phenoxazine (tCO) (X = O) in hybridization with guanine (A, C, D). The structure of pyrrolo-dC (E).
Pyrrolo-dC as Cytosine Analogue: In another study, a new highly fluorescent base analogue of cytosine, pyrrolo-dC (1.39), (Figure 1.10) has been introduced to characterize the transcription bubble in elongation complexes of T7 RNA
polymerase.26a-b Pyrrolo-dC has excitation and emission maxima at 350 nm and 460 nm, respectively, which, like the previously described analogues, allows selective excitation in the presence of native nucleic acid bases and proteins. This base analogue can pair with guanine and like 2-AP, pteridine and hydrocarbon base analogues, shows significant quenching of fluorescence when incorporated into single and double stranded DNA. The quenching can be used to monitor local melting of the G:C base pairs in a DNA helix and can serve as a complementary probe to 2-AP, which reports on melting of AT base pairs.26c
Xanthosine Analogue: The base analogue of the rare base xanthosine (5-aza-7- deazaxanthine) (1.38) has been synthesized and reported by Benner et al. (Figure 1.11).27 This base was designed based on the supposition that the rare tRNA constituent wyosine carries the 5-aza-7-deazapurine substructure, and it is this structure that makes the base fluorescent. When excited at 250 nm, this base analogue displays two emission maxima at 410 nm and 580 nm.
O HO
HO N
N N
N H H
H H
O OH
OH N
X N
N O
O H
H
O OH HO N N
N O
O H
H
X = H, OH
1.42 (xanthosine analogue) 5-aza-7-deazapurine 2'-deoxyriboside 1.40 1.41
2'-deoxyxanthosine (X =N)
Figure 1.11: Structure and hydrogen bonding property of xanthosine analogue.