Synthesis of novel derivatives of 5-hydroxymethylcytosine and 5-formylcytosine as tools for epigenetics.

In this work we present for the first time the synthesis of novel 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) derivatives that can be used as tools in the emerging field of epigenetics for deciphering chemical biology of TET-mediated processes.

Introduction

Epigenetic modifications play a crucial role in cell differentiation and cell development [1]. They control gene expression through several mechanisms such as non-coding RNAs, histone modifications (acetylation, methylation, phosphorylation, etc.) [2], and DNA methylation [3-7]. The latter takes place at the C-5 position of the cytosine moiety in CpG islands establishing the so called 5th base: 5-methylcytosine (5mC), a well-known epigenetic mark that correlates with gene silencing [8]. Recently, conversion of the 5mC moiety to 5-hydroxymethylcytosine (5hmC) and to higher oxidation products such as 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by the action of ten-eleven-translocation (TET) enzymes was discovered [9-13]. The TET proteins are identified as 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenases [10,14]. Whereas the DNA methylation is a densely studied field, its reverse process has not yet been deciphered. In trying to understand DNA demethylation several mechanisms involving new cytosine-modified bases as intermediates have been proposed (Scheme 1). (1) The most widely accepted pathway includes iterative oxidation of 5mC catalyzed by TET enzymes followed by removal of 5fC and 5caC by thymine DNA glycosylase (TDG). Excision of 5fC and 5caC generates an abasic site, which is further repaired resulting in replacement of 5mC with unmodified cytosine (C) [15-18]. (2) The second alternative scenario still remains controversial [19-20]. It links the oxidative action of TET enzymes, the subsequent deamination of 5hmC to 5-hydroxymethyluracil (5hmU) by cytidine deaminases AID (activation-induced cytidine deaminase) or APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) with the base excision repair (BER) machinery [15-1721]. (3) Among other putative demethylation mechanisms is the direct dehydroxymethylation of 5hmC to cytosine by action of DNA methyltransferases (DNMT). This enzymatic process was observed in vitro, whether it also works in vivo is yet to be elucidated [15,22]. (4) Lastly, decarboxylation of the 5caC by an unknown decarboxylase excluding action of BER should also be considered [15,23]. This variety of demethylation pathways might indicate that different tissues utilize different demethylation pathways [1,24].

Scheme 1

Proposed steps for DNA demethylation (for details see text).

While DNA methylation is usually associated with gene repression [8,25], active demethylation seems to allow cells to unblock silenced genes aiming at epigenetic reprogramming of their genetic material [26]. Current accepted models propose that 5hmC could be involved in epigenetic modulation of gene activity. In fact, 5hmC was discovered also in embryonic stem cells and seems to play a decisive role in their self-renewal process [27]. Interestingly, the levels of 5hmC in several cancer types are strongly reduced relative to the corresponding normal tissue around the tumor [28]. To gain deeper insights into the chemical biology of DNA demethylation pathways further exploration of the TET-mediated processes is necessary. Analogues of 5hmC with substituents preventing formation of 5fC and 5caC species could serve as useful tools for ongoing investigations in this emerging field.

Results and Discussion

Herein, we describe the synthesis of compounds with the general formula I which represents modified cytidine analogues bearing a secondary alcohol at position C-5 of cytosine. Additionally, a synthesis of 3,6-dihydrodeoxycytidine derivatives of general formula II is presented (Figure 1).

Figure 1

Structures of the synthesized compounds.

We chose the known aldehyde 1 [29] (prepared from commercially available 2’-deoxycytidine) as a starting material for the envisioned transformations (Scheme 2). To the best of our knowledge, the addition of organometallic compounds (organolithium and organomagnesium, etc.) to aldehyde 1 is not described in the literature. Compound 1 was readily converted to 5hmC analogues 2a–e by treatment with various Grignard reagents (methylmagnesium bromide, THF, 0 °C → room temperature, or vinylmagnesium bromide, THF, 0 °C → room temperature) and organolithium reagents (lithium (trimethylsilyl)acetylide, THF, −40 °C → −20 °C or lithium phenylacetylide, THF, −78 °C → −50 °C) (Scheme 2). These alcohols were obtained as a mixture of diastereomers in yields ranging from 43% to 96% (Table 1). Compound 2b was isolated in moderate yield of 43% due to the cleavage of the TMS-group during the reaction resulting in formation of derivative 2e with a yield of 26%. The obtained derivatives 2a–d were further treated with Olah’s reagent and pyridine in EtOAc at room temperature or HF·triethylamine complex [30] in DCM at 0 °C to afford the deprotected 2’-deoxycytidine analogues 3a–d as mixtures of diastereomers in yields of 60–75%.

Scheme 2

Synthesis of the 2'-deoxycytidine analogues.

Table 1

Yields and ratios of diastereomeric alcohols 2a–e and 3a–d.

Entry	2/3	Yield [%]	Ratioa
1	2a, R = methyl	96	1.1:1
2	2b, R = (TMS)ethynyl	43	1.9:1
3	2c, R = phenylethynyl	68	1.2:1
4	2d, R = vinyl	77	1.1:1
5	2e, R = ethynyl	26	1.2:1
6	3a, R = methyl	75	n.d.
7	3b, R = ethynyl	60	n.d.
8	3c, R = phenylethynyl	72	1.1:1
9	3d, R = vinyl	73	n.d.

aDetermined by 1H NMR; n.d. = not determined.

Next, we synthesized the N-4-protected cytidine derivatives 4 and 5 by treatment of aldehyde 1 with β,β,β-trichloro-tert-butoxycarbonyl chloride (TCBocCl) [31] in the presence of pyridine in DCM (Scheme 3). The reaction of 4 with Grignard (methylmagnesium bromide, THF, 0 °C → room temperature, or vinylmagnesium bromide, THF, 0 °C → room temperature) and organolithium reagents (lithium (trimethylsilyl)acetylide, THF, −60 °C → −50 °C or lithium phenylacetylide, THF, −78 °C) afforded derivatives 6a–c and carbamates 7a–c as mixtures of diastereomers (Table 2). It should be mentioned that upon storage at room temperature derivatives 6a–c undergo slow intramolecular cyclization to the corresponding carbamates 7a–c. The reaction of aldehyde 4 and vinylmagnesium bromide yielded directly carbamate 7d.

Scheme 3

Reactions of TCBoc-protected aldehydes 4 and 5 with organometallic reagents.

Table 2

Yields and ratios of diastereomers 6a–c, 7a–d, 8a–e and 9a–d.

Entry	6/7/8/9	Yield [%]	Ratioa
1	6a, R = methyl	28	n.d
2	6b, R = (TMS)ethynyl	42	2:1
3	6c, R = phenylethynyl	42	1.6:1
4	7a, R = methyl	35	1.9:1
5	7b, R = (TMS)ethynyl	30	1.1:1
6	7c, R = phenylethynyl	30	1.4:1
7	7d, R = vinyl	69	2.3:1
8	8a, R = methyl, R1 = H	38	2.4:1
9	8b, R = (TMS)ethynyl, R1 = H	80	3.2:1b
10	8c, R = phenylethynyl, R1 = H	71	1.1:1
11	8d, R = vinyl, R1 = H	37	2.6:1
12	8e, R = vinyl, R1 = TCBoc	17	5.7:1
13	9a, R = methyl	40	2.4:1
14	9b, R = (TMS)ethynyl	77	–
15	9c, R = phenylethynyl	44	1:1
16	9d, R = vinyl	61	2.6:1

aDetermined by 1H NMR; bpure epimers were isolated by HPLC; n.d. = not determined.

Surprisingly, the reaction of derivative 5 bearing a N-(TCBoc)2 group with organometallic compounds (methylmagnesium bromide, THF, 0 °C → room temperature, lithium (trimethylsilyl)acetylide, THF, −50 °C, lithium phenylacetylide, THF, −78 °C → −50 °C, vinylmagnesium bromide, THF, 0 °C) afforded 3,6-dihydrodeoxycytidine derivatives 8a–e as mixtures of diastereomers. In case of 8b the diastereomers were separated by HPLC. Products arising from addition of the organometallic reagents to the aldehyde group (1,2-addition) were not observed. The formation of compounds 8a–d can be explained assuming a Michael-type reaction of aldehyde 5 with organometallic reagents, subsequent isomerisation of the double bond followed by removal of one TCBoc group during the reaction and work-up as shown in Scheme 4.

Scheme 4

Proposed mechanism for the formation of 3,6-dihydrodeoxycytidine derivatives 8a–d (M = Li, Mg).

Finally, cleavage of the TCBoc group was achieved by the action of the 10% Cd–Pb couple [32] on compounds 8a–d in THF and 1.0 M aq NH4OAc to provide derivatives 9a–d (Scheme 3).

Conclusion

In summary, the reaction of 5fC derivatives 1, 4, and 5 with organometallic reagents (RMgBr, RLi) was investigated and enabled the synthesis of novel derivatives of 5-hydroxymethylcytosine and 5-formylcytosine: whereas aldehydes 1 and 4 afforded cytosine derivatives 2a–e, 6a–c and 7a–d, the reaction of derivative 5 yielded 3,6-dihydrodeoxycytidine derivatives 8a–d which subsequently after removal of the TCBoc group afforded derivatives 9a–d. These new nucleobase modified 2’-deoxycytidine analogues can be used in the synthesis [29,33-34] of modified DNA oligomers for further studies of the TET-mediated processes which are of great importance in the emerging field of epigenetics. In addition they could find application as novel antivirals and/or as antimetabolites [35-36]. The majority of the obtained compounds contain functionalized side chains thus allowing further manipulations.

Experimental details and analytical data of all synthesized compounds.