ISSN 1349-4848

number148

Contr ibution :- A Synthetic Biology Approach to the Expansion of

the Genetic Alphabet: Molecular Design of

Unnatural Base Pairs of DNA

Ichiro Hirao Michiko Kimoto Nucleic Acid Synthetic Biology Research Team, RIKEN SSBC

New Produc ts I nformation :- A Valuable Synthetic Intermediate for 2-Substituted

Adenosine

- Indicator Nucleoside of DNA Methylation

- A Unique and E�cient Ni Catalyst for Cross-Coupling

Reactions

- Useful DMAP Analogs

- Reagent for Introduction of the TTMSS Group

2

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CONTENTS

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Synthetic biology is a new research area, and its key objective is to create new biological systems by introducing non-natural (unnatural) components. Here, as one of the synthetic biology approaches, we describe studies of the expansion of the genetic alphabet of DNA by creating artificial base pairs (unnatural base pairs). Several unnatural base pairs that function as a third base pair in replication, transcription, and/or translation have recently been developed and are being utilized in a wide range of applications.

1. Introduction

In 2010, Craig Venter’s group reported the creation of a new Mycoplasma bacterium containing an artificially synthesized genome with 1.08 M bases.1 This achievement was the culmination of the coordinated efforts of chemists and biologists through a 5-year study. The generation of the initial cells required great effort, but once the artificial cells were created, they proliferated in media like natural cells. Thus, the artificially designed cells can be reproduced with reasonable costs and used as a biological factory to synthesize useful proteins and other materials. This re-design of an existing biological system is an example of a synthetic biology

approach.2 Another type of synthetic biology approach is the creation of a new biological system, constructed with newly designed biological components. In this approach, new, artificial components are developed to serve a certain purpose, and they function alongside the natural components in a biological system. The new components are created by repeated “proof of concept” experiments. A prototype component is designed based on a concept or an idea, and then it is improved according to the results from physical and biological experiments. Here, we describe this type of synthetic biology approach, through the creation of unnatural base pairs toward the expansion of the genetic alphabet of DNA.3-8

2. Development of unnatural base pairs

The genetic information of terrestrial life is encoded in DNA as a sequence comprising the four different bases, adenine (A), guanine (G), cytosine (C) and thymine (T), as alphabetical letters. In duplex DNA molecules, A selectively pairs with T, and G pairs with C. This base pairing rule is fundamental for the genetic information flow through replication, transcription, and translation. Thus, introducing an artificially created base

RNA

DNA

YTP Natural base NTPs

dXTP, dYTP Natural base dNTPs

Unnatural amino acid

tRNA

Protein

X C G

Met Phe Lys

Y

Y X

A A A A A A T T T T

T T C C C

C C G G G

G G

A A A U U U C C G G G

Unnatural amino acid

Figure 1. Expansion of the genetic alphabet by an unnatural base pairThe complementarity of the natural A-T (A-U in RNA) and G-C base pairs is a principle mechanism of genetic information flow. Introduction of an unnatural base pair (X-Y) into DNA provides a new biotechnology, allowing the site-specific incorporation of functional components into nucleic acids and proteins.

A Synthetic Biology Approach to the Expansion of the Genetic Alphabet: Molecular Design of Unnatural Base Pairs of DNA

Ichiro Hirao and Michiko Kimoto

Nucleic Acid Synthetic Biology Research Team, RIKEN SSBC

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pair into DNA could increase the genetic alphabet and expand the genetic information, providing a new biotechnology capable of the site-specific incorporation of new components into nucleic acids and proteins (Figure 1).9 In addition, recent physical and biological studies on replication and transcription, using artificial, unnatural bases, have revealed novel molecular interactions and biological reaction mechanisms, which had not been observed in conventional analyses using standard biomolecules with only the natural components. Furthermore, artificially increasing the genetic alphabet of a biological system is a formidable challenge for chemists. The most important issue is that the unnatural base pair (X–Y) functions as a third base pair with highly exclusive selectivity; namely, X selectively pairs with Y, alongside the A–T and G–C pairs in the biological system (Figure 2). In replication, DNA polymerase binds to a partially double-stranded DNA fragment between a primer and a template strand. Subsequently, a nucleoside 5′-triphosphate (dNTP, substrate) is imported into the polymerase–DNA complex. When the substrate base correctly pairs with its partner base

in the template, then the oxygen atom of the 3′-hydroxy group of the primer attacks the α position of the triphosphate in the substrate. This results in the formation of the phosphodiester bond between the primer and the imported substrate, and the release of pyrophosphoric acid (pyrophosphate) as a leaving group. After the incorporation of the correct substrate within the primer, the DNA polymerase slides along the template DNA strand, and the incorporation of the next substrate occurs. The selectivity of the natural base pairing in replication is extremely high. For example, in the case of the Klenow fragment of Escherichia coli DNA polymerase I, an incorrect base substrate is incorporated once every 10,000 times,10 which means that the selectivity of the natural base pairing by the Klenow fragment is 99.99% per replication.

3. Natural Base Pairs: A–T and G–C

To create an unnatural base pair, we need to understand why the natural A–T and G–C base pairs exhibit high selectivity

3

3

5

5

Figure 2. Replication mechanism involving an unnatural base pairDNA polymerase binds to the partially double-stranded DNA between a primer and a template DNA. The substrate, dYTP, is imported into the protein-DNA complex. When the Y base correctly pairs with the X base in the template, the 3′-hydroxy group of the primer DNA attacks the α position of the substrate phosphate, resulting in the formation of the phosphodiester bond and the release of the pyrophosphoric acid.

ribose ribose ribose ribose

A T G C

1 2 3

4

5 6 7

8 9

1 2 3

4

5 6 7

8 9

1 2 3

4 5 6 1 2

3

4 5 6

10.7-11.0 Å

： important proton-donor residues for polymerase recognition

10.7-11.0 Å

Figure 3. The natural A-T and G-C base pairsSince the distances between the glycoside positions of the pairing bases are always around 11 Å, DNA forms several types of regular double-helices. The Kool group has shown the importance of the shape complementarity between pairing bases in replication.

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in replication, in terms of their chemical, physical, and biological aspects. In the A–T and G–C pairings, purine bases (A or G) pair with pyrimidine bases (T or C), with two hydrogen bonds for A–T and three for G–C (Figure 3). Accordingly, the distances between the glycosidic bonds of each pairing base are always 10.7–11.0 Å, and the duplex DNA molecules adopt regular double-helical structures. Hydrogen bonds are formed between a proton donor residue and a proton acceptor atom or residue. Between the A–T and G–C pairs, the hydrogen bond patterns differ from each other, and thus A always pairs with T, and G always pairs with C in the regular helical structures. However, recent studies have revealed that hydrogen bond formation between pairing bases is not necessary for correct base pairing in replication. In 1995, Eric Kool and his colleagues designed and synthesized a hydrophobic, unnatural Z–F pair, in which the shapes of Z and F mimic those of A and T, respectively (Figure 4).11-13 For the Z base, the 1- and 3-nitrogens in A were replaced with carbons, and the 6-amino group was replaced with a methyl group. For the F base, the 3-imino group in T was replaced with a C-H group, and the 2- and 4-keto groups were replaced with fluorine atoms. In general, the proton acceptor ability of a fluorine atom is around ten times lower than that of a keto group. Kool’s group chemically synthesized DNA templates containing Z or F at a specific position, as well as triphosphates of their nucleosides (dZTP and dFTP), as substrates for replication experiments in vitro. They showed that the Klenow fragment of Escherichia coli DNA polymerase I efficiently incorporated both dFTP and dTTP into DNA opposite Z or A in the templates with similar efficiencies, and also incorporated both dZTP and dATP opposite F or T. In contrast, the incorporation efficiencies of dCTP opposite Z and dGTP opposite F were very low. Thus, the hydrophobic, unnatural Z–F pair also works in replication and is compatible with the A–T pair, suggesting that the shape complementarity between pairing bases is more important than the hydrogen-bonding complementarity in replication.14 For recognition by polymerases, the 3-nitrogens of A and G, and the 2-keto groups of C and T are necessary as hydrogen-bond acceptors oriented toward the minor groove, to interact

with specific amino acid residues in polymerases (Figure 3).15 For example, the substrates of pyrimidine analogs lacking the 2-keto groups are not incorporated into DNA in PCR (Polymerase Chain Reaction).16 Kool’s Z base also lacks the hydrogen acceptor atom corresponding to the 3-nitrogen of purines, and thus, they newly developed the Q base, which has a nitrogen at this position (Figure 4).17 The Klenow fragment replicated the Q–F pair with higher efficiency than the Z–F pair. Other factors, such as the hydrophobicity of bases, the dipole moments of pairing bases, the stacking interactions among neighboring bases, and the CH–π interactions18, are also important for the base pair selectivity in replication. With these points in mind, various types of unnatural base pairs have been developed thus far. In the following sections, we introduce representative unnatural base pairs and their developmental processes.

4. Hydrophilic unnatural base pairs by Benner’s group

In the late 1980s, Steven Benner and his colleagues reported four types of unnatural base pairs bearing different hydrogen-bonding geometries, which were unlike those of the A–T and G–C pairs.19, 20 A representative one is the base pair between 6-amino-2-ketopurine (isoG) and 2-amino-4-ketopyrimidine (isoC), which are the structural isomers of G and C, respectively (Figure 5).19 Benner’s group demonstrated that the isoG–isoC pair works complementarily in replication by the Klenow fragment. In addition, the isoG substrate (isoGTP) was incorporated into RNA opposite isoC in DNA templates by T7 RNA polymerase.21 Furthermore, in 1992, the isoG–isoC pair was applied to an in vitro translation system, allowing the site-specific incorporation of a non-standard amino acid (3-iodotyrosine) into a peptide, using a short, chemically synthesized mRNA containing isoC and a 3-iodotyrosyl tRNA containing isoG.22

These pioneering studies by Benner’s group attracted much attention to the genetic expansion system using unnatural base pairs. However, these unnatural base pairs still had some

A T G C

Q F Z F

1

2

： important proton-donor residues for polymerase recognition

ribose ribose ribose ribose

ribose ribose ribose ribose

Figure 4. Kool’s unnatural base pairsThe Kool group synthesized an unnatural Z-F pair, mimicking the shapes of the natural A-T pair. The non-hydrogen-bonded Z-F pair functioned in replication with high selectivity, showing the importance of the shape complementarily, rather than hydrogen bonding, between pairing bases in DNA polymerase reactions.

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shortcomings. For example, the isoG base undergoes keto-enol tautomerization in solution, and the enol form of isoG pairs with T (Figure 5).21 Besides the isoG problem, the 2-amino group of isoC, corresponding to the 2-keto position of the natural bases, reduces the interaction with polymerases.21 In addition, the nucleoside of isoC is unstable in aqueous solution, and 50% of the isoC nucleoside triphosphate is decomposed under neutral conditions in water at room temperature after four days.23 Although the introduction of a methyl group to position 3 of the isoC base, corresponding to position 5 of the natural pyrimidine bases, improved the stability, the methyl-isoC ribonucleoside is still susceptible to the epimerization of its β-glycoside bond, from the β- to α-form. Recently, Benner’s group addressed this problem by introducing a nitro group to position 3 of isoC,24 as described below. In 2005, Benner’s group solved the problem of the isoG keto-enol tautomerization by using a modified T base, 2-thiothymine (TS), instead of T (Figure 5), for the isoG–isoC pair system.25 The large size of the sulfur atom of TS prevents the pairing with the enol form of isoG, but it still pairs with a without steric repulsion. This strategy is similar to Kool’s concept of shape fitting between pairing bases, as well as the idea of the unnatural base pair reported by Harry Rappaport in 1988,26 who used a modified guanine base, 6-thioguanine, as a new base. The unnatural isoG–isoC pair functions together with the A–TS pair in PCR: the selectivity of the isoG–isoC pair reached 98% per replication, while the isoG–isoC selectivity without the help of TS was about 93% per replication.25 However, 98% unnatural base pairing selectivity is not sufficient for replication, and the retention rate of the unnatural base pair in 20-cycle PCR amplified DNA fragments becomes about 67% (0.9820 = 0.67). Thus, for the practical use of unnatural base pairs in replication, more than 99% selectivity per replication is required. In 2007, Benner’s group improved their unnatural base

pair system and developed the P–Z pair, which functions in PCR amplification without the use of the A–TS pair (Figure 5).24 Unlike isoG, the P base does not undergo the undesirable tautomerization in solution. The nitro group of the Z base prevents the epimerization of its nucleoside derivatives, due to the deprotonation of the imino group of the Z base. Although the selectivity of the P–Z pair in PCR was 97.5% at that time, the group recently reported improved selectivity to about 99.8% under optimized PCR conditions.27

5. Hydrophobic unnatural base pairs by Romesberg’s group

Kool’s pioneering experiments with the non-hydrogen-bonded base pairs generated interest in hydrophobic base pairs as unnatural base pair candidates. Floyd Romesberg and his colleagues developed numerous hydrophobic unnatural base pairs and examined their abilities in replication systems. In 1999, they reported a hydrophobic, isoquinoline derivative, PICS (Figure 6), which pairs self-complementarily in double-stranded DNA fragments with high thermal stability.28 In addition, the PICS substrate was enzymatically incorporated into DNA opposite PICS in templates by the Klenow fragment. Unfortunately, after the incorporation of the PICS substrate opposite PICS, the primer extension paused. This is because the shape of the PICS–PICS pair is too large for accommodation within the regular DNA double helix, in which the PICS bases stack on each other, and does not allow polymerase recognition. Therefore, they exhaustively designed and synthesized other hydrophobic, unnatural base pairs and examined their abilities in replication.29-35

In 2009, they succeeded in developing the hydrophobic, unnatural base pairs 5SCIS–MMO2 and 5SCIS–NaM (Figure 6), which function as a third base pair in PCR

A T G C

1

2

2

2

3

A TS

isoGenol TS isoGenol T isoG isoC

Z P

ribose ribose

ribose ribose ribose ribose

ribose ribose

ribose ribose

ribose ribose ribose ribose

Figure 5. Benner’s unnatural base pairsThe Benner group developed unnatural base pairs, such as isoG-isoC and Z-P, with different hydrogen bond donor-acceptor patterns from those of the A-T and G-C pairs. The P-Z pair functions in PCR amplification. Although the selectivity of the isoG-isoC pair in replication is not high because of the keto-enol tautomerization, the isoG-isoC pair can be used in PCR by using A-TS in place of the A-T pair.

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amplification.36, 37 As compared to the previous PICS–PICS pair, the shape complementarity of these base pairs was improved. The best fidelity of the 5SCIS–NaM pair in PCR reached 99.8%, although it depended on the sequence contexts around the unnatural base pair. These hydrophobic base pairs also function in transcription by T7 RNA polymerase.38 The group also engineered DNA polymerases by an evolutionary engineering technique, and employed mutated polymerases to solve the polymerase pausing problem when using the PICS–PICS pair in replication.39

6. A series of unnatural base pairs by Hirao’s group

Our group has also been studying unnatural base pairs since 1997. After struggling through lots of ‘proof of concept’ experiments, we developed our first unnatural base pair, between 2-amino-6-dimethylaminopurine (x) and 2-oxopyridine (y), in 2001 (Figure 7).40 The x–y was designed by combining two concepts: Benner’s hydrogen-bonding patterns, which differ from those of the natural base pairs, and a steric hindrance effect. The x–y pair has two hydrogen bonds, and the 2-aminopurine moiety of x may pair with T through a similar hydrogen bonding interaction as that with y. Thus, we added a bulky dimethylamino group to position 6 of x, to exclude the x–T pairing by steric hindrance between the 6-dimethylamino group and the 4-keto group of T. In contrast to T, y has a hydrogen atom, instead of a keto group. The x–y pair functions in transcription with high selectivity, and T7 RNA polymerase incorporated the triphosphate substrate of y (yTP) into RNA, opposite x in DNA templates, with more than 95% selectivity. Furthermore, we improved the shape complementarity of the unnatural base pair by replacing the dimethylamino group of x with a thienyl group. Thus, we developed 2-amino-6-thienylpurine (s) as the pairing partner of y (Figure 7).41 The planarity of the thienyl group excludes the mispairing with T more efficiently than the dimethylamino group of x. In addition, the planarity increased the stacking ability of the s base with neighboring bases in DNA strands. The selectivity and

efficiency of the s–y pair in transcription were greatly improved, as compared to those of the x–y pair, and not only yTP but also several modified yTPs, such as biotin- and fluorophore-linked y bases, were incorporated into RNA, opposite s in DNA templates, by T7 RNA polymerase.42-44 In 2002, by combining the specific transcription involving the s–y pair and an in vitro E. coli translation system, we succeeded in the site-specific incorporation of a non-standard amino acid, 3-chlorotyrosine, into a protein.41 Thus, the s–y pair functions as a third base pair in in vitro transcription and translation systems. We sought to improve our unnatural base pairs further. One major obstacle was the insufficient selectivity of the s–y pair for replication. After 10 cycles of PCR with a DNA fragment containing the s–y pair, around 40% of the unnatural base pair was replaced with the natural base pairs. The selectivity of the s–y pair is ~95% per replication (40% of replacement after 10-cycle PCR: 1−0.9510 = ~0.4), and thus, more than 99% selectivity is required for unnatural base pairs in replication, in which ~90% (0.9910 = ~0.90) of an unnatural base pair could be retained in its amplified DNA after 10 cycles of PCR. Therefore, we strictly refined the shape complementarity between the pairing bases and designed a five-membered ring nucleobase analog, imidazoline-2-one (z),45 instead of y with the six-membered ring, as the pairing partner of s (Figure 7). In addition, we removed the atoms and residues involved in hydrogen-bonding interactions between the pairing bases from the s–z pair,46 and developed a hydrophobic, unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]-pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) (Figure 7, see Appendix for chemical syntheses).47 We added an aldehyde group to the Pa base, as a proton acceptor for interactions with polymerases. The p rob lem wi th the Ds–Pa pa i r i s t ha t s e l f -complementary Ds–Ds pairing also competitively occurred in replication. As also found with Romesberg’s PICS–PICS pair, the extension paused after the Ds incorporation opposite Ds. Fortunately, we found that a modified triphosphate substrate, the γ-amidotriphosphate, of Ds was selectively incorporated opposite Pa, but showed greatly decreased incorporation opposite Ds.47 After the modified substrate is incorporated, the DNA has the native phosphodiester linkage, because the

PICS

1’

1

PICS

5SICS MMO2 5SICS NaM

2

ribose ribose

ribose ribose ribose ribose

Figure 6. Romesberg’s unnatural base pairsThe Romesberg group developed PICS, the first replicable self-pair. Then, they screened an unnatural hydrophobic base library, and discovered the highly selective 5SICS-MMO2 and 5SICS-NaM pairs. They also studied polymerase mutations for enhanced incorporation of the PICS-PICS pair.

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7

x

1

2

y

4

s y x T

3

5

s z Ds Pa

Ds Pn Ds Px A Pn

ribose ribose

ribose ribose ribose ribose ribose ribose

ribose ribose

ribose ribose ribose ribose

ribose ribose

Figure 7. Hirao’s unnatural base pairsWe developed several unnatural base pairs by combining the designed concepts of hydrogen-bonding patterns, shape complementarity with steric hindrance and electrostatic repulsion, and hydrophobicity. The Ds-Px pair exhibits high fidelity and efficiency in PCR amplification.

amidopyrophosphate moiety of the substrate is removed as a leaving group during polymerization. For PCR amplification, we also used the γ-amidotriphosphate of A, besides Ds, to reduce the misincorporation of dATP opposite Pa. In 2006, we successfully performed highly selective PCR amplification involving the Ds–Pa pair using the γ-amidotriphosphates of A and Ds, in which the selectivity of the Ds–Pa pairing reached more than 99% per replication. As a serendipitous mistake, we accidentally synthesized the γ-amidotriphosphates by treating the triphosphate synthesis intermediates, the cyclic 5′-triphosphates, with concentrated ammonia. We further improved the Ds–Pa pair, and replaced the aldehyde group of Pa with a nitro group, thus designing 2-nitropyrrole (Pn) (Figure 7).48 The nitro group of Pn can reduce the misincorporation of A opposite Pa, by the electrostatic repulsion between the oxygen of the nitro group and the 1-nitrogen of A. In addition, we added a propynyl group to position 4 of the Pn base, and developed 2-nitro-4-propynylpyrrole (Px) as a pairing partner of Ds (Figure 7).49 The propynyl group increases the hydrophobicity, which strengthens the interaction with polymerases. Thus, the Ds–Px pair functions in PCR without the aid of the γ-amidotriphosphates. DNA fragments containing the Ds–Px pair were amplified 108-fold by 40 cycles of PCR, and more than 97% of the Ds–Px pair was retained in the amplified DNA. The selectivity of the Ds–Px pair reached more than 99.9% per replication, which is the highest selectivity among the unnatural base pairs reported thus far.50 Diagnostic and therapeutic applications using the Ds–Px pair are in progress. In addition to these unnatural base pairs mentioned here, we also reported unique base pairs with fluorophore or quencher abilities and their applications.

7. Conclusion

The synthetic biology of unnatural base pairs has rapidly advanced over the past 20 years, and various types of unnatural base pairs were developed (Figure 8).4, 51-54 These unnatural base pairs have been applied to site-specific fluorescent labeling,42, 55 immobilization,43, 47 specific detection,56-60 and structural analyses61-63 of DNA and RNA molecules. Although the applications of unnatural base pairs to translation are still being developed,22, 41 it is only a matter of time until a protein synthesis technology is developed for the site-specific incorporation of non-standard amino acids into proteins. The previous unnatural base pair studies were limited to in vitro experiments. However, we now have several types of unnatural base pairs with the potential for use in in vivo experiments. In the future, expanded genetic alphabets using unnatural base pairs will be applied to cells, such as Venter’s artificial cell, to efficiently produce artificial proteins and to trace target gene expression in the cell. We look forward to further advancements in this area, toward next generation biotechnologies.

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Appendix 1

Appendix 2

Synthesis of nucleoside derivatives of DsReagents and abbreviations: (a) dichlorobis(triphenylphosphine)palladium, 2-(tributylstannyl)thiophene, DMF; (b) palladium on carbon, sodium borohydride, ethanol, ethylacetate; (c) formic acid; (d) NaH, 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride, CH3CN; (e) NH3, methanol; (f) 4,4’-dimethoxytrityl chloride, pyridine; (g) 2-cyanoethyl tetraisopropylphosphor-diamidite, tetrazole, CH3CN; (h) acetic anhydride, pyridine, then dichloroacetic acid, dichloromethane; (i) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, dioxane, pyridine, tributylamine, bis(tributylammonium)pyrophosphate, DMF, then I2/pyridine, water, NH4OH (for triphosphate), I2/pyridine, NH4OH (for γ-amidotriphosphate); (j) tetra-O-acetyl-β-D-ribofuranose, chloroacetic acid. Tol: toluoyl, DMT: 4,4’-dimethoxytrityl, Ac: acetyl.

Synthesis of nucleoside derivatives of PaReagents and abbreviations: (a) NaH, 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride, CH3CN; (b) NH3, methanol; (c) 4,4’-dimethoxytrityl chloride, pyridine; (d) 2-cyanoethyl N,N-diisopropylaminochlorophosphoramidite, diisopropylethylamine, THF; (e) 1,8-bis(dimethylamino)naphthalene, POCl3, trimethyl phosphate, then tributylamine, bis(tributylammonium)pyrophosphate, DMF; (f) NaH, CH3CN, then 2,3,5-tri-O-benzyl-D-ribofuranosyl chloride; (g) BBr3, dichloromethane. Tol: toluoyl, DMT: 4,4’-dimethoxytrityl, Ac: acetyl.

xA T

Im-No Na-ON

A xT

Im-ON Na-NO

ribose ribose ribose ribose

ribose ribose ribose ribose

Figure 8. Other unnatural base pairs

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36. D. A. Malyshev, Y. J. Seo, P. Ordoukhanian, F. E. Romesberg, J. Am. Chem. Soc. 2009, 131, 14620–14621.

37. D. A. Malyshev, D. A. Pfaff, S. I. Ippoliti, G. T. Hwang, T. J. Dwyer, F. E. Romesberg, Chemistry 2010, 16, 12650–12659.

38. Y. J. Seo, S. Matsuda, F. E. Romesberg, J. Am. Chem. Soc. 2009, 131, 5046–5047.

39. A. M. Leconte, L. Chen, F. E. Romesberg, J. Am. Chem. Soc. 2005, 127, 12470–12471.

40. T. Ohtsuki, M. Kimoto, M. Ishikawa, T. Mitsui, I. Hirao, S. Yokoyama, Proc. Natl. Acad. Sci. USA 2001, 98, 4922–4925.

41. I. Hirao, T. Ohtsuki, T. Fujiwara, T. Mitsui, T. Yokogawa, T. Okuni, H. Nakayama, K. Takio, T. Yabuki, T. Kigawa, K. Kodama, K. Nishikawa, S. Yokoyama, Nat. Biotechnol. 2002, 20, 177–182.

42. R. Kawai, M. Kimoto, S. Ikeda, T. Mitsui, M. Endo, S. Yokoyama, I. Hirao, J. Am. Chem. Soc. 2005, 127, 17286–17295.

43. K. Moriyama, M. Kimoto, T. Mitsui, S. Yokoyama, I. Hirao, Nucleic Acids Res. 2005, 33, e129.

44. I. Hirao, Biotechniques 2006, 40, 711–715.45. I. Hirao, Y. Harada, M. Kimoto, T. Mitsui, T. Fujiwara, S.

Yokoyama, J. Am. Chem. Soc. 2004, 126, 13298–13305.46. T. Mitsui, A. Kitamura, M. Kimoto, T. To, A. Sato, I.

Hirao, S. Yokoyama, J. Am. Chem. Soc. 2003, 125, 5298–5307.

47. I. Hirao, M. Kimoto, T. Mitsui, T. Fujiwara, R. Kawai, A. Sato, Y. Harada, S. Yokoyama, Nat. Methods 2006, 3, 729–735.

48. I. Hirao, T. Mitsui, M. Kimoto, S. Yokoyama, J. Am. Chem. Soc. 2007, 129, 15549–15555.

49. M. Kimoto, R. Kawai, T. Mitsui, S. Yokoyama, I. Hirao, Nucleic Acids Res. 2009, 37, e14.

50. R. Yamashige, M. Kimoto, Y. Takezawa, A. Sato, T. Mitsui, S. Yokoyama, I. Hirao, Nucleic Acids Res. in press.

51. J. Gao, H. Liu and E. T. Kool, J. Am. Chem. Soc. 2004, 126, 11826–11831.

52. N. Minakawa, S. Ogata, M. Takahashi, A. Matsuda, J. Am. Chem. Soc. 2009, 131, 1644–1645.

53. S. Hikishima, N. Minakawa, K. Kuramoto, Y. Fujisawa, M. Ogawa, A. Matsuda, Angew. Chem. Int. Ed. Engl. 2005, 44, 596–598.

54. C. Kaul, M. Muller, M. Wagner, S. Schneider, T. Carell, Nat. Chem. 2011, 3, 794–800.

55. M. Kimoto, T. Mitsui, S. Yokoyama, I. Hirao, J. Am. Chem. Soc. 2010, 132, 4988–4989.

56. C. B. Sherrill, D. J. Marshall, M. J. Moser, C. A. Larsen, L. Daude-Snow, S. Jurczyk, G. Shapiro, J. R. Prudent, J. Am. Chem. Soc. 2004, 126, 4550–4556.

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57. S. C. Johnson, D. J. Marshall, G. Harms, C. M. Miller, C. B. Sherrill, E. L. Beaty, S. A. Lederer, E. B. Roesch, G. Madsen, G. L. Hoffman, R. H. Laessig, G. J. Kopish, M. W. Baker, S. A. Benner, P. M. Farrell, J. R. Prudent, Clin. Chem. 2004, 50, 2019–2027.

58. P. Sheng, Z. Yang, Y. Kim, Y. Wu, W. Tan, S. A. Benner, Chem. Commun. 2008, 5128–5130.

59. S. Hoshika, F. Chen, N. A. Leal, S. A. Benner, Angew. Chem. Int. Ed. Engl. 2010, 49, 5554–5557.

60. R. Yamashige, M. Kimoto, T. Mitsui, S. Yokoyama, I. Hirao, Org. Biomol. Chem. 2011, 9, 7504–7509.

61. M. Kimoto, M. Endo, T. Mitsui, T. Okuni, I. Hirao, S. Yokoyama, Chem. Biol. 2004, 11, 47–55.

62. M. Kimoto, T. Mitsui, Y. Harada, A. Sato, S. Yokoyama, I. Hirao, Nucleic Acids Res. 2007, 35, 5360–5369.

63. Y. Hikida, M. Kimoto, S. Yokoyama, I. Hirao, Nat. Protoc. 2010, 5, 1312–1323.

Introduction of the authors :

Ichiro Hirao Team Leader, RIKEN, Systems and Structural Biology Center, Nucleic Acid Synthetic Biology Research Team, and CEO, TagCyx Biotechnologies

[Education and employment] 1983 Ph.D., Department of Chemistry, Faculty of Science, Tokyo Institute of Technology; 1984-1993 Assistant Professor, Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo (Supervisor: Prof. Kin-ichiro Miura); 1993-1996 Associate Professor, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1995-1997 Associate Scientist, Department of Chemistry, Indiana University (Supervisor: Prof. Andrew Ellington); 1997-2001 Group Leader, ERATO, Japan Science and Technology Corporation (Collaborator: Prof. Shigeyuki Yokoyama); 2002-2006 Professor, Research Center for Advanced Science and Technology, The University of Tokyo; 2007-present President & CEO, TagCyx Biotechnologies; 2007-present Visiting Professor, Graduate School of Chemical Sciences and Engineering, Hokkaido University; 2008-present Team Leader, Systems and Structural Biology Center, RIKEN[Specialties] Synthetic Biology, Organic Chemistry, Molecular Biology, Evolutional Biology

Michiko Kimoto Researcher, RIKEN, Systems and Structural Biology Center, Nucleic Acid Synthetic Biology Research Team

[Education and employment] 1999-2002 Junior Research Associate, RIKEN, Cellular Signaling Laboratory; 2002 Ph.D., Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo (Supervisor: Prof. Shigeyuki Yokoyama); 2002-2006 Research Associate and 2006-2008 Research Scientist, RIKEN, Genomic Sciences Center; 2008-present Research Scientist, RIKEN, Systems and Structural Biology Center, RIKEN[Specialties] Molecular Biology and Biochemistry

No.148 No.148

11

TCI Related Compounds

Chapter 4. Hydrophilic unnatural base pairs by Benner’s group

Chapter 6. A series of unnatural base pairs by Hirao’s group

Others

Chapter 5. Hydrophobic unnatural base pairs by Romesberg’s group

Isoguanine100mg[I0370]

3-Bromo-5-methyl-2-pyridone1g, 5g

[B3350]

2-Bromo-3-methoxynaphthalene1g, 5g

[B3403]

5-Methyl-2-thiouracil10g, 25g[M0994]

Pyrrole-2-carboxaldehyde5g, 25g[P1246]

N

NH

NH

N

O

NH2

NH

BrCH3

O

Br

OCH3

NH

NH

CH3

O

S

NH

C H

O

1,8-Bis(dimethylamino)-naphthalene1g, 5g, 25g

[B1018]

Dichloroacetic Acid25g, 500g [D0308]

Palladium 5% on Carbon (wetted with ca. 55% Water) 5g, 25g [P1490]Palladium 10% on Carbon (wetted with ca. 55% Water) 5g, 25g [P1491]Sodium Borohydride 100g, 500g [S0480]Sodium Hydride (60%, dispersion in Paraffin Liquid) 100g [S0481]

Nucleosides, Nucleotides & Related Reagentshttp://www.tcichemicals.com/eshop/en/ap/category_index/00301/

Bis(triphenylphosphine)-palladium(II) Dichloride

1g, 5g, 25g [B1667]

4,4'-Dimethoxytrityl Chloride5g, 25g [D1612]

2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one

5g, 25g [C1210]

Trimethyl Phosphate25g, 500g [P0271]

2-Cyanoethyl N,N,N',N'-Tetraisopropylphosphordiamidite

1g, 5g [C2228]

1H-Tetrazole5g, 25g [T1017]

NN CH3

CH3CH3

CH3

OH

O

Cl

Cl

P3

P3

PdCl

Cl

CCH3O

OCH3

Cl

OP

O

O

Cl

O

P

OCH3

OCH3CH3O

OCH2CH2CN

PN N

CH(CH3)2

CH(CH3)2

(CH3)2CH

(CH3)2CH

N

NH

N

N

No.148

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No.148

Figure 2. Bifunctional catalysts bearing protic chelate amine ligands.

A Valuable Synthetic Intermediate for 2-Substituted Adenosine

I0759 2-Iodoadenosine (1) 200mg, 1g, 5g

Adenosine derivatives play important roles in metabolic actions in various cells, and show antiviral and anticancer activities. 2-Iodoadenosine 1 is a valuable synthetic intermediate for the preparation of 2-substituted adenosine derivatives. Here are two typical adenosine derivatives syntheses using cross-coupling reactions. Matsuda et al. have reported a synthesis of 2-alkynyladenosines using a palladium-catalyzed cross-coupling reaction, and evaluation of their pharmacological activities.1) Zhao and Baranger have reported a synthesis of 2-alkenyladenosines using the Heck reaction.2)

Typical Procedure1c)

2-Iodoadenosine (393 mg), CuI (9.5 mg), bis(triphenylphosphine)palladium(II) dichloride (36 mg), triethylamine (0.16 mL) and ethynylbenzene (123 mg) in DMF (7 mL) are heated at 80 °C for 1 h. After 2-iodoadenosine is completely consumed, the reaction mixture is concentrated to dryness under reduced pressure. The residue is dissolved in CHCl3, and H2S gas is introduced to the solution (~30 s) followed by N2 gas. The suspension is filtered through a Celite pad and washed with CHCl3. The combined filtrate is concentrated to dryness in vacuo, and the residue is purified by silica gel column chromatography (eluent: MeOH–CHCl3) to give 2-(phenylethyn-1-yl)adenosine (373 mg, 97%).

References1) Cross-coupling reaction with terminal alkynes a) A. Matsuda, M. Shinozaki, T. Miyasaka, H. Machida, T. Abiru, Chem. Pharm. Bull. 1985, 33, 1766. b) A. Matsuda,

T. Ueda, Nucleosides Nucleotides, 1987, 6, 85. c) T. Abiru, T. Miyashita, Y. Watanabe, T. Yamaguchi, H. Machida, A. Matsuda, J. Med. Chem. 1992, 35, 2253.

2) Cross-coupling reaction with terminal alkenes (Heck reaction) Y. Zhao, A. M. Baranger, J. Am. Chem. Soc. 2003, 125, 2480.

Related CompoundsB1667 Bis(triphenylphosphine)palladium(II) Dichloride 1g 5g 25gA1424 Palladium(II) Acetate 1g 5gT1024 Tris(2-methylphenyl)phosphine 5g 25g

O

OH

HO

OH

N

N N

N

NH2

I

O

OH

HO

OH

N

N N

N

NH2

CC

1

PdCl2(PPh3)2

DMF, Et 3N (1.2 eq.)80 °C, 1 h

CuI (5 mol%)(10 mol%)

C CH (1.2 eq.)

O

OH

HO

OH

N

N N

N

NH2CH2CH CH2

Pd(OAc)2

CH3CN, Et3N (2.74 eq.)80 °C, overnight

(1.2 eq.)

(1.02 eq.)

(3.3 eq.)

Y. 53%

1c)

2)

R

R

R = Hydroxyalkyl Hydroxycycloalkyl Cycloalkyl, Ph

Y. 55-97%

P(o-Tolyl )3

No.148 No.148

13

Indicator Nucleoside of DNA Methylation

D3610 2'-Deoxy-5-methylcytidine (1) 100mg, 500mg, 5g

Biological consequences of DNA methylation are indicators of epigenetic effects in plants and animals. 2’-Deoxy-5-methylcytidine, formed by the enzymatic methylation of cytosine residues in CpG (Cytosine-phosphate-Guanine) sites, is a minor component found in most eukaryotic DNA which plays a central role in the regulation of gene expression. Recently, methylation occurring by methyl radicals generated by tumor promoters has been reported.1) Oxidation of 2’-deoxy-5-methylcytidine has been of interest in connection with the damage of DNA.2) In the detection of DNA methylation levels in cells, the DNA is hydrolyzed to nucleosides and 2’-deoxy-5-methylcytidine is analyzed by HPLC and LC-MS.3)

References1) Cytosine C-5 methylation by methyl radicals generated by tumor promoters K. Kawai, Y.-S. Li, M.-F. Song, H. Kasai, Bioorg. Med. Chem. Lett. 2010, 20, 260.2) Oxidation studies of 2’-deoxy-5-methylcytidine a) T. Itahara, Chem. Lett. 1991, 1591. b) T. Itahara, T. Yoshitake, S. Koga, A. Nishino, Bull. Chem. Soc. Jpn. 1994, 67,

2257. c) C. Bienvenu, J. R. Wagner, J. Cadet, J. Am. Chem. Soc. 1996, 118, 11406. d) T. Umemoto, A. Okamoto, Org. Biomol. Chem. 2008, 6, 269.

3) Analyzed of 2’-deoxy-5-methylcytidine in cells a) M. Mizugaki, K. Itoh, T. Yamaguchi, S. Ishiwata, T. Hishinuma, S. Nozaki, N. Ishida, Biol. Pharm. Bull. 1996, 19, 1537. b)

Z. Liu, S. Liu, Z. Xie, W. Blum, D. Perrotti, P. Paschka, R. Klisovic, J. Byrd, K. K. Chan, G. Marcucci, Nucleic Acids Res. 2007, 35, e31. c) A. A. Magaña, K. Wrobel, Y. A. Caudillo, S. Zaina, G. Lund, K. Wrobel, Anal. Biochem. 2008, 374, 378. d) M. Münzel, D. Globisch, T. Brückl, M. Wagner, V. Welzmiller, S. Michalakis, M. Müller, M. Biel, T. Carell, Angew. Chem. Int. Ed. 2010, 49, 5375.

O

OH

HO N

N

NH2

O

CH3

1

A Unique and Efficient Ni Catalyst for Cross-Coupling Reactions

B3534 Bis(tricyclohexylphosphine)nickel(II) Dichloride (1) 1g

The Ni-based catalyst bis(tricyclohexylphosphine)nickel(II) dichloride [NiCl2(PCy3)2] (1) has been reported as a unique and efficient transition metal catalyst for cross-coupling reactions (e.g. Suzuki–Miyaura, Kumada, etc.), which are efficient methods for the construction of C–C bonds.

References1) For Suzuki-Miyaura cross-coupling a) D. Zim, V. R. Lando, J. Dupont, A. L. Monteiro, Org. Lett. 2001, 3, 3049. b) L. Xu, B.-J. Li, Z.-H. Wu, X.-Y. Lu, B.-T.

Guan, B.-Q. Wang, K.-Q. Zhao, Z.-J. Shi, Org. Lett. 2010, 12 , 884.2) For Kumada cross-coupling B.-T. Guan, X.-Y. Lu, Y. Zheng, D.-G. Yu, T. Wu, K.-L. Li, B.-J. Li, Z.-J. Shi, Org. Lett. 2010, 12, 396.3) Mechanism of Ni-catalyzed selective C–O bond activation in cross-coupling Z. Li, S.-L. Zhang, Y. Fu, Q.-X. Guo, L. Liu, J. Am. Chem. Soc. 2009, 131, 8815.

OTsB(OH)2

Ph

Dioxane, 130 °C, 14-60 h

Y. 98 %(1.5 eq.)

1a)

+1 (3 mol%), PCy3 (2 eq.), K3PO4 (12 mol%)

P

Ni

P

Cl

Cl

3

3

No.148

14

No.148

Useful DMAP Analogs

A1854 9-Azajulolidine (1) 1g

9-Azajulolidine (1) is a strong acylation catalyst, which displays remarkably enhanced catalytic activity toward the acylation of sterically hindered alcohols compared to 4-dimethylaminopyridine (DMAP) and other analogs.1)

Because of its high electron-rich character, 1 is also useful as an effective auxiliary ligand for promoting post-Ullmann coupling reactions leading to the construction of C(aryl)-heteroatom bonds.2) It can be applied to the synthesis of triarylamines, which are important hole-transporting materials in many organic optoelectronic devices.

Typical Procedure: C–N bond formationCuI (9.5 mg, 0.05 mmol), base (2.0 mmol), aryl iodide (1.0 mmol) and aryl amine (1.2 mmol) are added to a two-neck flask equipped with a septum. The flask is evacuated and back-filled with argon. Then toluene (4 mL) and 9-azajulolidine (1.0 mL, 0.1 mmol, 0.1 M in toluene) are added by a syringe at room temperature. The reaction mixture is heated at 110 °C for 40 h. The reaction mixture is allowed to cool to room temperature, water (20 mL) is added, and the mixture is extracted with CH2Cl2 (15 mL × 3). The combined organic solution is dried over MgSO4 and concentrated in vacuo to yield the crude product, which is purified by chromatography on silica gel.

Reference1) Enhancing the catalytic activity of 4-(dialkylamino)pyridines by conformational fixation a) M. R. Heinrich, H. S. Klisa, H. Mayr, W. Steglich, H. Zipse, Angew. Chem. Int. Ed. 2003, 42, 4826. b) S. Singh, G.

Das, O. V. Singh, H. Han, Tetrahedron Lett. 2007, 48, 1983.2) Application of 9-azajulolidine to post-Ullmann reactions K.-T. Wong, S.-Y. Ku, F.-W. Yen, Tetrahedron Lett. 2007, 48, 5051.

+toluene, 110 °C

CuI (5 mol%), baseCH3 I XHAr

X = NH, NR, O, S

CH3 X Ar

1 (10 mol%) 2)

N

N

Yield (%)base

96

80*

98*

XHAr

Br SH Cs2CO3

*CuI (10 mol%), 1 (20 mol%)

OH

CH3

CH3

Cs2CO3

t-BuONaNH

NH

CH3 71t-BuONa

No.148 No.148

15

Reagent for Introduction of the TTMSS Group

C2411 Chlorotris(trimethylsilyl)silane (1) 5g

Chlorotris(trimethylsilyl)silane (1) is a reagent for the introduction of tris(trimethylsilyl)silyl group (TTMSS group) as a protecting group. 1 reacts with primary or secondary alcohols in the presence of DMAP to give the corresponding TTMSS ethers in high yields.1) TTMSS ethers are stable to Grignard reagents, oxidation and acidic conditions. They are also stable to CsF, conditions under which cleavage of silyl ethers is often observed. TTMSS ethers can be cleaved with photolysis (254 nm) or by the action of TBAF. Furthermore, in the Mukaiyama cross-aldol reaction2) and the [2+2] cyclization with acrylates3) using silyl enol ethers, the corresponding products can be obtained in high yields with high diastereoselectivity by the introduction of TTMSS group.

Typical Procedure: Protection of the Alcohol1)

To a stirring solution of the alcohol (1 mmol) and 4-dimethylaminopyridine (DMAP) (1.20 mmol) in CH2Cl2 is added a solution of chlorotris(trimethylsilyl)silane (1–1.2 mmol) in CH2Cl2 (1 M). The solution is stirred overnight at room temperature under N2. Water is added and the aqueous layer is extracted with CH2Cl2. The organic layer is dried over anhydrous sodium sulfate, filtered and the solvent is removed under reduced pressure. Separation by flash chromatography gives the products in 80–90% yields.

References1) The tris(trimethylsilyl)silyl group: an alcohol protecting group a) M. A. Brook, C. Gottardo, S. Balduzzi, M. Mohamed, Tetrahedron Lett. 1997, 38, 6997. b) M. A. Brook, S. Balduzzi, M.

Mohamed, C. Gottardo, Tetrahedron 1999, 55, 10027.2) Tris(trimethylsilyl)silyl-governed aldehyde cross-aldol cascade reaction M. B. Boxer, H. Yamamoto, J. Am. Chem. Soc. 2006, 128, 48.3) Remarkable tris(trimethylsilyl)silyl group for diastereoselective [2+2] cyclizations M. B. Boxer, H. Yamamoto, Org. Lett. 2005, 7, 3127.

R

R'OH

Si(CH3)3Si

Si(CH3)3

Si(CH3)3

Cl

1 (1.0 eq.)

DMAP (1.2 eq.)R

R'O Si

Si(CH3)3

Si(CH3)3

Si(CH3)3

R

R'OH + silylated products

CH2Cl2, rt, overnight

1)

hν / MeOH or TBAF

Y. 80-90%

O

n-BuLi

rt, 16 hO

OR

TTMSSOOR

O

Al catalyst (3 mol%)

O

Ph

Ph

AlNTf2

2

Y. >80% (>99% d.e. )

CH2Cl2, −40 °C, 4 h

O

OBn

CH3

HNTf2 (0.05 mol%)

CH2Cl2, −78 °C to rt, 1 h

TTMSSO OTTMSS

OBn

CH3

Y. 61% (pure diastereomer shown)

O

1

rt, 8 h

TTMSSO

2)

3)

Al catalyst =

silyl enol ether

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