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Photochemical & Photobiological Sciences View Article Online

DOI: 10.1039/C8PP00161H

Xiaoxu Li,a,b Ece Erturk,a,b Xin Chen,a,b Shiv Kumar,a,b Cheng Guo,a,b Steffen Jockusch,c James J. Russo,a,b Timothy H. Bestor,*d Jingyue Ju*a,b,e

Published on 12 June 2018. Downloaded by Tufts University on 6/15/2018 12:59:11 PM.

a

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027; b Department of Chemical Engineering, Columbia University, New York, NY 10027; c Department of Chemistry, Columbia University, New York, NY 10027; d Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY 10032; e Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, NY 10032 *Corresponding Authors: [email protected] and [email protected]

Abstract Epigenetic information is encoded in the mammalian genome in the form of cytosines methylated at the 5 position. While cytosine methylation has multiple biological effects, including gene regulation and silencing of integrated viral DNA, currently available methods for mapping methylation sites genome-wide have severe shortcomings. For instance, the gold standard bisulfite sequencing approach suffers from the use of harsh reaction conditions resulting in DNA cleavage and incomplete conversion of unmethylated cytosine to uracil. We report here on a new photochemical method in which a DNA (cytosine-5)-methyltransferase can be used to covalently attach reactive functionalities which upon irradiation at ~350 nm initiate photoinduced intramolecular reactions that convert methylated C to T analogues. We synthesized a model compound, a cinnamyl ether-containing cytidine derivative, and demonstrated its conversion to a thymidine analogue using mild conditions and a DNA-compatible wavelength (~350 nm), enabled by the use of a triplet sensitizer, thioxanthone. Transfer of a cinnamyl ether or comparable reactive functionality from an AdoMet analog to cytosine followed by the use of this photoconversion method would require only small amounts of DNA and allow complete methylation profiling on both long and short read sequencing platforms. Introduction DNA methylation is an epigenetic signal in mammalian genomes that regulates expression of imprinted genes, transposons, and genes on the inactive X chromosome in females.1 Methylation primarily takes place on cytidines (C) in symmetrical CpG sequences.1 The human genome contains ~29,000,000 CpG dinucleotides (5’-CG-3’

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Photochemical & Photobiological Sciences Accepted Manuscript

Photochemical Conversion of a Cytidine Derivative to a Thymidine Analog via [2+2]-Cycloaddition


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dinucleotides), each of which can exist in the methylated or unmethylated states. Genomic methylation patterns are essential for cell viability,2, 3 and abnormal DNA methylation is observed in many diseases, most notably cancer.4 However, extant methods for whole genome methylation profiling remain expensive, inefficient, and inaccurate. Improved methods of methylation profiling are urgently needed. Advances in chemical and biological studies on methylation analysis5 gave birth to new methods for cytosine conversion to distinguish between methylated and unmethylated C’s in DNA. Research into DNA methyltransferases has produced a novel tool kit for chemical modification of the 5 position of unmethylated C’s in CpG sites followed by conversion of the residues to thymidines (T). Several mechanisms are known for C to T conversion. One is oxidative deamination of the 4-amino group of C in the presence of nitrous acid and alkyl nitrite.6 Alternatively, photo-irradiation can catalyze ring formation between a reactive group at the 5-position and the 4-amino group leading to a pyrido(2,3-d)pyrimidine nucleoside.7 The 5,6-double bond of C can undergo an addition reaction which interrupts the conjugated system resulting in deamination of the 4-amino group and formation of a T analog (as in bisulfite mediated methylation profiling of DNA8, 9). Moreover, photo-irradiation generates a 5,6-cyclobutane intermediate between an alkene and the 5,6-double bond via a [2+2]-cycloaddition, leading to the interruption of the conjugated system, and deamination at the 4-position.10 This chemistry has been applied to on-DNA conversion of C to T, in which a double bond containing T is positioned in close proximity to C with the aid of hybridization after which photoirradiation causes C to T conversion.11, 12 We are currently developing a new method that will identify the methylation status of CpG sites (Scheme 1). In the first step AdoMet analogues bearing sulfonium-linked functionalities rather than methyl groups will allow DNA (cytosine-5)-methyltransferases (DNA MTase) to transfer reactive functionalities (R) to the 5-position of C in unmethylated CpG sites.13-15 Transfer to 5-methylated Cs cannot occur. In the second step the covalently attached reactive functionalities (R) initiate photoinduced intramolecular reactions that convert Cs to T analogues. After conversion, DNA sequencing with all available platforms will unambiguously identify all unmethylated CpG sites as TpG dinucleotides; methylated CpG sites will be sequenced as CpG.

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In search of a reactive functionality (R; Scheme 1) capable of efficient conversion of C to T, we synthesized the cytosine derivative 1 (Scheme 2), which contains a cinnamyl ether functionality at the 5-position as a model compound for testing the photoconversion step. Here we investigate the efficiency of photochemical conversion of this C-derivative to a T-derivative. NH2 O

N N

HO

O

O

1 OH

Scheme 2: Cinnamyl ether derivative of deoxycytidine (1).

Results and Discussion The cinnamyl ether substituted deoxycytidine derivative (1) shows absorption of light at wavelength below 300 nm, which is caused by an overlap of absorption of the cinnamyl and cytosine chromophores (Figure 1). However, irradiation at wavelengths of 300 nm and below is undesirable because of unwanted side reactions which damage DNA. Therefore, triplet sensitization with thioxanthone (TX) was performed. TX has a strong absorption at ~380 nm (Figure 1), a wavelength which is benign to DNA. Photoexcitation of TX leads to singlet excited states which undergo efficient intersystem crosses into the triplet state with quantum yields of 0.6 to 0.8.16 The triplet energy of TX (269 kJ/mol) is higher than the triplet energy of the cinnamyl ether chromophore (260 kJ/mol) but significantly lower than the cytosine chromophore (334 kJ/mol).17 Therefore, efficient triplet sensitization will occur with the cinnamyl ether chromophore, but not the cytosine chromophore. To examine this difference in sensitization experimentally, the bimolecular rate constants for quenching of TX triplet states by these two chromophores were determined by laser flash photolysis using cinnamyl alcohol (2) and deoxycytidine (dC) as model compounds for the two chromophores. After pulsed laser excitation at 355 nm, TX shows a strong triplet absorption at 625 nm18 with a lifetime of ~30 µs under our experimental conditions. Pseudo-first order quenching kinetics at various quencher concentrations yielded the rate constants for TX triplet quenching by 2 and dC of 7.8 × 109 and 0.2 × 109 M-1s-1, respectively (Figure 2). The high rate constant for the cinnamyl chromophore ensures efficient triplet sensitization, whereas the 40 times lower rate constant for dC will greatly reduce unwanted side reactions. 3



Scheme 1: Enzymatically aided transfer of reactive functionality (R) to unmethylated CpG sites of DNA followed by photochemical conversion of modified Cs to T analogues.



kq2 = (7.8±0.2) x 109 M-1s-1 [2] (µM) OH

50 100

0.04

150 200 250

0.02

kobs625nm (106 s-1)

0

0.06

2

NH 2 N N HO

O

O

dC OH

kqdC = (2±1) x 108 M-1s-1

0.00 0

1

2

3 4 time (µs)

5

6 [quencher] (µM)

Figure 2: Left: Decay traces of the transient absorbance of TX in the triplet state monitored at 625 nm after pulsed laser excitation (355 nm, 5 ns pulse width) in argon saturated acetonitrile solutions in the absence and presence of various concentrations of 2 (0 to 0.25 mM). Right: Determination of the triplet quenching rate constant. Pseudo-first order rate constant of the decay of the triplet TX at various concentrations of 2 and dC.

To test for photochemical conversion of 1 to a deoxythymidine (dT) analog, deoxygenated acetonitrile solutions of 1 and TX were irradiated in a Rayonet reactor at 350 nm. The reaction was monitored by HPLC (Figure 3). While the concentration of the sensitizer (TX) remained unchanged during the course of the reaction, there was over 70% conversion of 1 to new photoproducts after 20 min irradiation. The major fraction at 4


Figure 1: Absorbance of 1 and TX in acetonitrile.

∆A625nm


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Absorbance @ 254 nm (AU)

0.35

before photolysis

0.30

NH 2 O

0.25

N 0.20

O

N

HO

O

S

O

TX

1

0.15

OH

0.10 0.05 0.00 0.00

Absorbance @ 254 nm (AU)


~26 min retention time was isolated and analyzed by APCI mass spectrometry, infrared spectroscopy (Figure S1, ESI), 1H-NMR (Figure S2, ESI) and 13C-NMR (Figure 4). Consistent with these analytical data, this photoproduct was identified as a cyclobutyl derivative of dT (4), either as straight (4a) or cross adduct (4b) as shown in blue at the bottom of Figure 3. Under these mild photolysis conditions, the cyclobutyl derivative of dT is the stable reaction product.

5.00

10.00

20.00

25.00

30.00

35.00

40.00

Major photoproduct

0.20

O

O

O

N HO

OH

N

or

0.05

HO

O

O OH

4a

50.

NH

O

O

45.00

20 min photolysis at ~350 nm

O

NH

0.15

0.10

15.00

4b

0.00 0.00

10.00

20.00

30.00

40.00

50.00

Retention time (min)

Figure 3: Top: HPLC analysis (monitored by UV absorbance at 254 nm) of aqueous solutions containing acetonitrile (65%), 1 (3 mM) and TX (100 µM) before photoirradiation. Bottom: analysis after 20 min photoirradiation with 350 nm light.

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Figure 4: 13C-NMR analysis of 1 (top) and isolated photoproduct 4 (bottom) in DMSOd3. Major shifts were observed for carbon atoms 5, 6, c, and d (marked in red). For simplicity, only the straight [2+2]-cycloadduct is shown.

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1. hν

O

O

NH 2

O

N 3TX*

N

+1 - TX

2.

HO

NH 2

*

3


O

3a

O

OH

HO

O OH

3b

O

5.

OH O

O

H2O pH 7

HO NH

N HO

NH

O

O

O OH

5

O OH

O N

O

4.

N

O

O

4a

O N

31*

N

NH 2

O

HO

4. H2O pH 7

O OH

[2+2]-cycloaddition

N N

HO

3.

NH O

4b

Scheme 3: Photochemical conversion of 2 to T analogues. The mechanism of photoconversion is shown in Scheme 3. (1) Photoexcitation of TX with ~350 nm light followed by intersystem crossing generates TX triplet states. (2) The TX triplet states are efficiently quenched by 2 as shown by laser flash photolysis (Figure 2) by triplet energy transfer to the cinnamyl chromophore of 1. (3) The triplet excited state of the cinnamyl double bond undergoes a [2+2]-cycloaddition reaction with the 5, 6 double bond of the cytosine moiety of 1. Cycloaddition may take place either in the straight form (3a) or as a cross-adduct (3b). (4) Because of the loss of the double bond in cytosine, oxidative deamination at the 4-position occurs spontaneously in aqueous solution (pH = 7)10 generating thymidine analogues 4a and/or 4b. (5) In the final step, cycloreversion would generate 5. Cycloreversion of similar dT derivatives have been reported in the literature.11, 12 Although the cyclobutyl derivatives 4a and 4b we generated in TX-sensitized photoconversion of 1 vary from the canonical base T, they bear all of the base pairing features of T.

Conclusions Photoproduct studies showed that the cinnamyl ether derivative of cytidine (1) can be efficiently converted to a T analogue with ~350 nm light using TX as triplet sensitizer. Triplet sensitization of this reaction is efficient as demonstrated by the high triplet quenching rate constant which is close to the diffusion limit. Our studies suggest that the cinnamyl ether substituent in conjunction with TX triplet sensitization should perform sufficiently as a reactive functionality (R; Scheme 1) that can convert C to T analogues for methylation profiling of DNA.

Experimental Section

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TX



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Synthesis of 1 was performed as outlined in Scheme 4. Deoxythymidine was purchased from TCI. All other chemicals were purchased from Sigma-Aldrich. 1H-NMR and 13CNMR spectra were recorded on Bruker 300 or 400 MHz and Bruker 75 or 100 MHz spectrometers, respectively, and reported as shifts in ppm from a methanol-d4, chloroform-d or DMSO-d6 internal standard (3.31, 7.26 and 2.50 ppm, respectively, for 1 H-NMR and 49.86, 77.36 and 39.51 ppm, respectively, for 13C-NMR). Data were reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets; coupling constant(s) in Hz). Mass spectra (MS) were obtained on an Advion Mass Spec mass spectrometer equipped with both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) probes.

Scheme 4: Synthesis of the cinnamyl ether derivative of deoxycytidine (1).

3'-5'-Bis-O-(tert-Butyldimethylsilyl)-5-Bromomethyl-Deoxyuridine (7): 3'-5'-bis-O-(tert-butyldimethylsilyl)-thymidine (6) was synthesized from deoxythymidine following a published procedure.19 In the following reaction step, to a solution of 3'-5'bis-O-(tert-butyldimethylsilyl)-thymidine (6) (1g, 2.124 mmol) in 100 ml benzene, 2,2'azobisisobutyronitrile (AIBN) (10 mg, 0.03 eq.) and N-bromosuccinimide (NBS) (654 mg, 3.67 mmol, 1.7 eq.) were added. The mixture was heated under reflux for 4h. After

8



Synthesis of 1:

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cooling to room temperature, the reaction mixture was concentrated to dryness under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate/hexane: 1/5-1/3) to give pure product (7) (0.862 g, 74%). 1H-NMR (Figure S3, ESI) (300 MHz, CDCl3) δ: 8.80 (s, 1H), 7.88 (s, 1H), 6.29 (t, 1H), 4.40 (m, 1H), 4.23 (q, 2H), 3.98 (d, 1H), 3.91-3.75 (dd, 2H), 2.36-2.28 (m, 1H), 2.05-1.96 (m, 1H), 0.95-0.89 (m, 18H), 0.14-0.08 (m, 12H)). 13C-NMR (Figure S4) (75 MHz, CD3OD) δ 161.83, 150.08, 139.61, 112.11, 85.61, 85.95, 72.68, 63.44, 41.25, 26.39, 26.14, 25.48, -4.24, 5.25; APCI-MS: calculated for C22H42BrN2O5Si2 [(M+H)+]: 549.17, found: 549.30 and 551.29. 3'-5'-Bis-O-(tert-Butyldimethylsilyl)-5-Cinnamyloxymethyl-Deoxyuridine (8): NaH (16 mg) was added to a solution of cinnamyl alcohol (2) (91.8 mg, 0.6843 mmol, 1.5 eq.) in anhydrous THF (5 ml), and the mixture was stirred for 0.5 h. Then the mixture was added dropwise into a solution of 3'-5'-bis-O-(tert-butyldimethylsilyl)-5bromomethyluridine (7) (250 mg, 0.456 mmol). The reaction mixture was stirred at room temperature for 16 h, and concentrated to dryness under reduced pressure. The residue was dissolved in DCM (2 ml) and was purified by silica gel column chromatography (elution with DCM to remove unreacted starting material, cinnamyl alcohol, followed by elution with ethyl acetate/hexane: 1/3-1/2) to yield the pure product 8 (136 mg, 49.6%). 1 H-NMR (Figure S5) (400 MHz, CDCl3) δ: 8.20 (s, 1H), 7.68 (s, 1H), 7.47-7.31 (m, 5H), 6.65 (d,1H), 6.33 (m,1H), 6.27 (t, 1H), 4.42 (m, 1H), 4.27 (s, 2H), 4.21 (t, 2H), 3.85 (m, 1H), 3.79-3.76 (m, 2H), 2.31-2.23 (m, 1H), 2.04-2.00 (m, 1H); 0.90 (s, 18H), 0.08 (s, 12H); 13C-NMR (Figure S6) (75 MHz, CDCl3) δ 162.75, 150.34, 138.65, 136.98, 135.87, 133.42, 128.91,128.13, 126.92, 126.00, 112.19, 88.30, 85.67, 72.65, 72.04, 64.78, 63.37, 41.77, 26.33, 26.13, -4.27, -5.11; APCI-MS: calculated for C31H51N2O6Si2 [(M+H)+]: 603.32, found: 603.50. 3'-5'-Bis-O-(tert-Butyldimethylsilyl)-5-Cinnamyloxymethyl-Deoxycytidine (10) 1,2,4-triazole (312 mg, 453 mmol, 20 eq.) was suspended in acetonitrile (8 ml), to which phosphoryl chloride (P(O)Cl3) (86.7 mg, 51.8 µl, 0.5655 mmol, 2.5 eq.) was added dropwise and the mixture was stirred at 0 °C for 10 min. After addition of triethylamine (TEA) (502.6 mg, 692 µl, 4.98 mmol, 22 eq.) over 3 min, the mixture was stirred for a further 20 min, then a solution of 3'-5'-bis-O-(tert-butyldimethylsilyl)-5cinnamyloxymethyl-deoxyuridine (8) (136 mg, 0.226 mmol) in acetonitrile (5 ml) was added. The reaction solution was stirred at room temperature for 1h, then TEA (184.7 µl) and H2O (48.6 µl) were added and the mixture was stirred for 10 min. The reaction mixture containing 9 was concentrated to dryness under reduced pressure. 1,4-Dioxane (5 ml) and concentrated ammonium hydroxide (NH4OH) (3 ml) were added to the residue and the mixture was stirred for 1 h. The reaction mixture was concentrated again to dryness under reduced pressure, acetyl acetate was added to dissolve the residue and the

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organic phase was washed with brine (3 × 40 ml). The organic phase was concentrated to dryness and the residue was purified by silica gel column chromatography (ethyl acetate) to give pure product 10 (84.1mg, 62%). 1H-NMR (Figure S7) (400 MHz, CDCl3) δ: 7.75 (s, 1H), 7.41-7.28 (m, 5H), 6.65 (d,1H), 6.33 (m,1H), 6.25 (t, 1H), 4.36 (m, 1H), 4.34 (s, 2H), 4.16 (t, 2H), 3.95 (m, 1H), 3.77-3.72 (q, d, 2H), 2.49-2.42 (m, 1H), 2.02-1.93 (m, 1H); 0.90 (s, 18H), 0.08 (s, 12H); APCI-MS: calculated for C31H52N3O5Si2 [(M+H)+]: 602.34, found: 602.73. 5-Cinnamyloxymethyl-Deoxycytidine (1) 3'-5'-bis-O-(tert-butyldimethylsilyl)-5-cinnamyloxymethyl-deoxycytidine (84 mg, 0.14 mmol) was dissolved in THF (4 ml), and TBAF solution (1 M in THF) (0.56 ml, 4 eq.) was added while stirring. The reaction mixture was stirred for 2 h. Afterwards, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography purification (DCM/MeOH 10/1-4/1) to yield pure product 1 (45.9 mg, 88%). 1H-NMR (Figure S8) (300 MHz, DMSO-d6) δ 7 .88 (s, 1H), 7.46 – 7.22 (m, 6H), 6.64 (s, 1H), 6.59 (s, 1H), 6.42 – 6.33 (m, 1H), 6.15 (t, J = 6.6 Hz, 1H), 5.18 (d, J = 4.2 Hz, 1H), 4.99 (t, J = 5.3 Hz, 1H), 4.28-4.24 (m and s, 3H), 4.12 (d, J = 3.7 Hz, 2H), 3.78 (q, J = 3.7 Hz, 1H), 3.57 (m, J = 5.1 Hz, 2H), 2.22 – 2.06 (m, 1H), 2.02 – 1.93 (m, 1H); 13C-NMR (Figure S9) (75 MHz, CDCl3) δ 165.33, 155.70, 141.78, 137.31, 132.23, 129.45, 128.45, 127.35, 127.18, 103.10, 88.11, 85.76, 71.13, 70.40, 66.57, 62.15, 41.26; APCI-MS: calculated for C19H24N3O5 [(M+H)+]: 374.41, found: 374.75. Photoirradiation and Analysis: For photoirradiation, 1 (3 mM) and TX (0.1 mM) were dissolved in an aqueous solution containing 65% acetonitrile. The solution was placed in a Kimble KIMAX borosilicate glass culture tube (75 mm × 10 mm) with a silicone cap and deoxygenated by purging with argon for 20 minutes. The samples were irradiated in a Rayonet reactor (Southern New England Ultraviolet Co.) equipped with 12 light tubes with emission centered at 350 nm. The progress of the photoreaction was monitored by HPLC (Waters), using a 15 cm x 4.6 mm, 3 µM silica-C18 column and an elution gradient: A 3 min; B 0% in A to 40% in 30 min; to 80% in 10 min; A: H2O, B: methanol. The main product fraction was collected and analyzed by APCl mass spectrometry, 1H-NMR, 13C-NMR, and FT-IR spectroscopy. Analysis of photoproduct 4 after photolysis of 1: 1 H-NMR (Figure S10) (300 MHz, DMSO-d6) δ 10.69 (s, 1H), 7.40 – 7.19 (m, 5H), 5.78 (dd, J = 9.7, 5.2 Hz, 1H), 4.93 (d, J = 4.0 Hz, 1H), 4.77 (t, J = 5.5 Hz, 1H), 4.33 (d, J = 8.4 Hz, 1H), 4.16 (d, J = 9.1 Hz, 1H), 4.03 (d, J = 8.9 Hz, 2H), 3.75 (d, J = 9.1 Hz, 1H), 3.64 (dd, J = 9.5, 5.6 Hz, 1H), 3.52 (d, J = 5.1 Hz, 1H), 3.43 (dq, J = 10.3, 4.9 Hz, 3H),

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3.16 (t, J = 5.7 Hz, 1H), 1.64-1.54 (m, 1H), 0.97-0.91 (m, 1H); 13C-NMR (Figure S11) (300 MHz, DMSO-d6) δ 171.10, 153.04, 139.44, 129.68, 129.10, 127.84, 86.13, 83.33, 75.46, 74.24, 71.51, 62.83, 53.23, 51.62, 51.28, 50.04, 37.10; APCI-MS found: 375.72, calculated for C19H23N2O6 [(M+H)+]: 375.39. Spectroscopy: IR spectra were obtained using a Nicolet Nexus 870 FT-IR spectrometer. UV-vis spectra were recorded on an Agilent 8453 spectrometer. Laser flash photolysis experiments employed the pulses from a Spectra Physics GCR-150-30 Nd:YAG laser (355 nm, 5 ns pulse width) and a computer-controlled system, as described previously.20 Solutions of the sensitizer (TX) were prepared at a concentration such that the absorbance was 0.3 at the excitation wavelength (355 nm) and deoxygenated by purging with argon for 20 minutes. Acknowledgements This work was supported by the National Institutes of Health (5R21HG009187-02). Electronic supplementary information (ESI) available: IR spectra, NMR spectra.

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DOI: 10.1039/C8PP00161H

Page 13 of 13


DOI: 10.1039/C8PP00161H

TOC: A cinnamyl ether containing cytidine derivative is photochemically converted


under mild conditions into a thymidine analog. This conversion can be employed for development of a new method for DNA methylation profiling.

hν

O O S

NH 2

O NH

O

N N N

HO

O

O

O OH

Cytidine derivative

HO

O OH

Thymidine analog

13


Graphical Contents Entry:

Supersonic Jet Studies of Fiwene Clustered with Water, Ammonia, and

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