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The 2-Thioanalogues of /RNA Components Derived from 5-Hydroxyuridine Andrzej J. Małkiewicz* and Barbara Nawrot Institute of Organic Chemistry, Technical University, Lodz. Zwirki 36, Poland Z. Naturforsch. 42b, 355-359 (1987); received June 30, 1986

Modification. /RNA Components, Modified Uridines, 2-Thiouridine Derivatives, Nucleosides Synthesis

The synthesis of 5-cyanomethoxy-2-thiouracil (5b) is described. The acid catalysed ethanolysis of 5b leads to 5-ethoxycarbonylmethoxy-2-thiouracil (5a). Condensations of 5-methoxy-2-thiouracil (5) and 5a with the sugar component 7 using the "silyl method" yield peracylated 5-methoxy-2-thiouridine (8) and 5-ethoxycarbonylmethoxy-2-thiouridine (8a), which under treatment with sodium methanolate/methanol afford title 5-methoxy-2-thiouridine (1) (s2mo5U) and 5-methoxycarbonylmethoxy-2-thiouridine (2) (s :mcmo5U), respectively.

Introduction

5-Methoxyuridine (mo5U) ( l a ) and 5-methoxycar-bonylmethoxyuridine (mcmo3U or mV) (2 a) occupy the "first position" of several /RNA anticodons ("wobble position", U34) and recognize not only A and G but also U as "third letter" of cognate codons [1, 2]. Recently Yokoyama et al. [3, 4] have pos-tulated post-transcriptional 2-thiation of U34 to be a main factor leading to the restriction in base pairing of such a modified unit with G and U.

HN

HO

0

x i HO OH

x = s 1 x = o 1a

O C H 3 HN

X^N

O C H 2 C O O C H 3

HO OH

x = s 2 x = 0 2a

Thus, considering the significance of the genetic code redundancy [5] one can expect 5-methoxy-2-thiouridine (1) and 5-methoxycarbonylmethoxy-2-thiouridine (2) to be unfunctional components in the formation of rRNA structures being capable of rec-ognizing several synonymous codons. In fact, although the majority of uridines located at the "wobble position" have their own 2-thio-counter-parts e.g.: mnm"U, s2mnm'U; mcm?U, s2mcnrU;

* Reprint requests to Dr. A. J. Matkiewicz.

Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0340 - 5087/87/0300 - 0355/$ 01.00/0

cmnnrU, s2cmnnrU; the nucleosides 1 and 2 have not been found yet in about 280 of known /RNA sequences [1].

However, whether the mentioned restriction in the base pairing of U34 results by the influence of the 2-thio substituent on the conformation of such modified uridines, or whether it is attributed to the direct of Mg2" ion mediated interaction of 5-sub-stituents with backbone is still under discussion [3, 4, 6 - 1 1 , 22],

It should be also recalled that, in response to phys-iological stress the structure-modifying apparatus of a cell can alter not only a level but also a nature of U34 modification [2, 12]. The reported new synthesis of the nucleosides 1 and 2 provides potential compo-nents of /RNAS sequence, models to study conforma-tional dynamics of modified uridines under physio-logical conditions [25] and useful substrates to extend investigations of nucleotides and oligonucleotides [3, 4],

Results and Discussion

The use of the "silyl method" [21] for the synthesis of modified uridines depends on the preparation of suitable heterobases. A number of "wobble uridine" heterobases have been synthesized employing the general approach, which consists of the condensation of type 4 "active esters" with thiourea in the presence of an alkaline catalyst [13—18].

In this way 5-methoxy-2-thiouracil (5) has been obtained in good yield [18]. A direct adaptation of this method for the synthesis of (5a) failed, as the condensation of the intermediate 4a with thiourea under various experimental conditions did not pro-vide 2-thiouracil derivatives not even in chromato-graphically detectable amounts.

356 A. J. Malkiewicz-B. Nawrot • The 2-Thioanalogues of rRNA Components

^COOC2H5 CH2

\ > C H 2 R

HC00C;HS> NQH/DME

R =-H 3 R=-COOC2H5 3a R = -CN 3b

HSC200Cs /0CH2R

S . - A Na 0 H

R=-H U R=-COOC?H5 U a R = -CN 4 B

0

S N H

1. NH2CSNH2/C2H5OH . reflux |

2. CH3COOH

R = - H 5 R = -COOC2HS 5 a R = - CN 5b

5b HCl / C 2 H 5 O H

reflux , 2.5 h

SCHEME 2

5a

As an alternative it has been shown that, the analogous reaction of 3 b leads to 5 b, which by acid catalysed alcoholysis can be transformed into the de-sired 2-thiouracil derivative 5 a. The compound 3 b was prepared in high yield by alkylation of sodium ethyl glycolate with bromoacetonitrile [23] in di-methoxyethane. Next, 3b was condensed with ethyl formate in the presence of sodium hydride (5 °C, D M E as solvent) to give 4 b. The crude enol 4 b was reacted with thiourea in refluxing ethanol. Unfortu-nately, after neutralization and evaporation of sol-vent 5-cyanomethoxy-2-thiouracil (5 b) did not pre-cipitate in cold water and the separation of the prod-uct mixture was accomplished using silica gel chromatography. The nitrile 5b and ester 5 a (as the partial ethanolysis product of 5 b) were eluated in 11% and 3% yield, respectively and their structures were confirmed by the spectral data. It is noteworthy that, the condensation of alternatively a-formylated ester 3b (at the nitrile group) with thiourea, leading to the respective derivative of cytosine, was not ob-served.

Ethanolysis of 5 b was performed by refluxing the substrate with a mixture of hydrochloric acid and ethanol. The ester 5a was purified by crystallization and its spectral data and t . l .c . mobility were in agreement with those, recorded for the previously isolated sample.

The S,0-disilylated heterobases 6 and 6a were prepared by the reaction of 5 and 5 a with trimethyl-silyl chloride in the presence of triethylamine [19].

Although several more effective silylating agents are in common use, this method has proved to be of

value for the synthesis of persilylated uracils, which are sensitive to ammonia or to heat [20].

The condensations of 6 and 6 a with peracylated sugar 7 were performed using the described proce-dure [14, 15] and the reaction mixtures were worked up according to the usually practiced method [14,15]. The N rribonucleosides 8 and 8a were isolated by means of silica gel chromatography in satisfactory yield as well as N^Nj-diribosides (ca. 5% yield) and Nrribosides (ca. 10% yield), which were eluated as faster and slower moving zones in comparison with N rregioisomers, respectively. The noticable amount of N3-regioisomers and N^Ns-diribosides results from the formation of the strong o complexes be-tween the heterobases 6 and 6 a having electron donating groups at their 5-positions and the catalyst employed [21],

The deprotection of the sugar moieties of 8 and 8 a combined with the transestrification of 8 a was car-ried out by treatment with 0,01 N sodium methano-late in methanol [13]. The crude products 1 and 2 were purified by crystallization in high yield and their structures confirmed by NMR and MS spectra. These data and the chromatographic mobilities of 2 in different solvents were identical to those obtained from authentic specimens prepared by the alterna-tive method [22].

Experimental

The melting points are uncorrected. UV spectra: Specord UV-VIS spectrometer. 'H NMR spectra: Bruker HX-72 instrument, tetramethylsilane was used as the internal standard. Electron impact mass

357 A. J. Malkiewicz-B. Nawrot • The 2-Thioanalogues of r R N A Components

CH3 I /

CH3-Si-S I

CH3/CH3

0-Si-CH3

J k J

CH3

R= - H 6 R = -COOC2HS 6A

0 Ac Bz

BzO OBz 7

Ac = CH3CO-Bz = C6H5CO-

SnClt CH 3CN. R.T.. 12h

HN

Bz -I^Uv.

O C H 2 R

8 (or8a I

BzO OBz

R = -H 8 R=-COOC2H5 8a

SCHEME 3

0.01 n MeONo/MeOH

48H R.T.

1 (or 2 )

spectra (MS) at 15 eV: LKB 2091 spectrometer, ab-brev. B-heterobase; s-sugar. The thin-layer chromatography (t.l.c.) was performed on Merck silica gel plates 60 F254 using the following systems (v/v): chloroform —methanol 9:1 (A); chloroform— methanol 85:15 (B); benzene—ethyl acetate 5:3 (C). The silica gel column chromatography was car-ried out on silica gel Merck 60 F (230—400 mesh). All evaporations were made under reduced pressure and bath temperature below 40 °C.

1. Ethyl cyanomethoxyacetate (3 b) To the stirred suspension of sodium hydride

(3.95 g, 0.16 mol) in anhydrous dimethoxyethane (DME, 80 ml) ethyl hydroxyacetate (17.1 g, 0.16 mol) was added dropwise. The stirring was con-tinued for 30 min, bromoacetonitrile (19.74 g, 0.16 mol) [24] was added, the reaction mixture re-fluxed for 6 h and left overnight at room tempera-ture. The precipitate was filtered off, the filtrate evaporated and the residue destilled to give 3 b (21.6 g, 90%) b.p. 98/15 mm.

NMR (CDC13): 4.26 (q., 7 = 7 Hz, 2H, - C H : - C H 3 ) , 4.22 (s., 2H, - C H . - C O ) , 4.17 (s., 2H, -CH 2 CN) , 1.25 (t., 7 = 7 Hz, 3H, - C H 2 - C H 3 ) .

IR (film): 2250 (—CN), 1740 ( -COOC 2 H 5 ) cm - 1 .

2. 5-Cyanomethoxy-2-thiouracil (5 b) To the stirred suspension of sodium hydride

(2.4 g, 0.1 mol) in DME (60 ml) the mixture of ethyl cyanomethoxyacetate 3b (14.3 g, 0.1 mol) and ethyl

formate (11.3 ml, 0.15 mol) was added dropwise at 0—5 °C. The stirring was continued for 2 h at room temperature and thiourea (5.23 g, 0.07 mol) in an-hydrous ethanol (250 ml) was introduced in one por-tion. The reaction mixture was refluxed for 7 h, the solvent evaporated and the heavy oil was dissolved in water (70 ml). The solution was acidified with acetic acid to pH = 3.5 and evaporated to dryness. The residue was dissolved in ethanol (200 ml), impurities filtered off and to the filtrate silica gel (10 g) was added. Ethanol was evaporated, silica gel co-evapo-rated with toluene (3x50 ml), suspended in chloroform and applied on silica gel column (60 g). The eluation was performed with chloroform — methanol 97:3 (v/v) to give three fractions (a, b, c).

a: 5a (600 mg, 3%) Rf = 0.53 (A). This sample exhibits Rf value and the spectral data identical to that recorded for the authentic specimen prepared by ethanolysis of 5 b.

b: 5b (2 g, 11%) Rf = 0.37 (A) m.p. 223-224 °C (from ethanol).

NMR (CD3SOCD3): 7.39 (s., H, H6), 4.92 (s., 2H, —CH2—).

UV (H ,0 ) : pH = 2, A max = 277; pH = 12, A max = 229, 263.

IR (KBr): 2280 ( - C N ) cm"1. MS: mle 183 (100%, M+) , 156 (1.5%, M - H C N ) ,

143 (27%, M - C H 2 C N ) . c: Thiourea m.p. 182 °C, Rf = 0.11 (A).

3. 5-Ethoxycarbonylmethoxy-2-thiouracil (5 a) A solution the nitrile 5b (2.59 g, 0.014 mol) in the

mixture of ethanol (50 ml) and concentrated hydro-

358 A. J. Malkiewicz-B. Nawrot • The 2-Thioanalogues of rRNA Components

chloric acid (10 ml) was refluxed for 3 h (progress of the reaction was monitored by 1.1. c.) and the solvent evaporated. The residue was co-evaporated with ethanol (3x40 ml) and applied on a silica gel column using the procedure described for the preparation of 3 b. The heterobase 5 a was eluated with chloroform — methanol 9:1 (v/v) in 60% yield (2.1 g) m.p. 169-170 °C (from ethanol).

NMR (CD3SOCD3): 7.50 (s.. H, H6), 4.95 ( s . ,2H, — O—CH?—), 4.47 (q., J = 1 Hz, 2H, - C H , C H 3 ) , 1.50 (t., J = 7 Hz, 3H, - C H 3 ) .

UV (H 2 0) : pH = 2, A max = 272; pH = 12, A max = 242, 268.

MS: mle (230, 100%, M+) , 157 (85%, M-COOC 2 H 5 ) , 128 (5%, M-OCH 2 COOC 2 H 5 ) .

4. S,0-Di-(trimethylsilyl)-5-methoxy-2-thiouracil (6) and S, 0-di-(trimethylsilyl)-5-ethoxycarbonyl-methoxy-2-thiouracü (6 a)

General procedure To the suspension of 5 [18] or 5 a (10 mmol) in

anhydrous benzene (150 ml) trimethylsilyl chloride (2.61 g or 3.04 ml, 24 mmol) in anhydrous benzene (30 ml) followed by triethylamine (2.43 g or 3.34 ml, 24 mmol) in benzene (30 ml) were added. The reac-tion mixture was vigorously stirred for 3 days at room temperature. The precipitate was filtered off, it was washed with anhydrous benzene (50 ml), the filtrates were combined and the solvent evaporated (all oper-ations were made under dry nitrogen). The residue was destilled on high vacuum (0.01 mm) to give: 6 (2.7 g, 90%) MS: mte 287 (10%, M - C H 3 ) , 6a (3.6 g, 82%) MS: mle 359 (5.4%, M - C H 3 ) .

5. 2' ,3' ,5'-Tri-O-benzoyl-5-methoxy-2-thiouridine (8)

Stannous tetrachloride (3.3 ml, 28 mmol) in an-hydrous acetonitrile (60 ml) was added dropwise to the stirred solution of l-0-acetyl-2,3,5-tri-0-ben-zoyl-/3-D-ribofuranose 7 (10.1 g, 20 mmol) and the silylated heterobase 6 (6.64 g, 22 mmol) in acetoni-trile (250 ml). The reaction mixture was left for 16 h at room temperature and acetonitrile evaporated to one third of the starting volume. The residue was poured into ice-cold 5% aqueous solution of sodium bicarbonate (400 ml) and the mixture extracted with chloroform (4x100 ml). The extracts were com-bined. washed with 10% aqueous solution of sodium chloride, water and dried. Chloroform was evapo-rated and the residue chromatographed on silica gel column using chloroform —methanol 98:2 (v/v) to give 8 (7.5 g, 62%) Rf = 0.59 (C).

NMR (CDClj): 8.21-7.20 (m., 15H. H-aromat.), 5.87 (br.s., H. H t ' ) , 3.25 (s., 3H, - C H 3 ) .

Analysis for C31H26N209S (602.54) Calcd C 61.80 H 4.40 N 4.05 S 5.31, Found C 61.37 H 4.12 N 3.95 S 5.43.

6. 2',3',5'-Tri-O-benzoyl-5-ethoxycarbonylmethoxy-2-thiouridine (8 a)

To the solution of 6a (2.4 g, 6 mmol) and 7 (3.3 g, 6.3 mmol) in acetonitrile (150 ml) stannous chloride (0.85 ml, 7.3 mmol) was added. The reaction mix-ture was worked up according to the procedure de-scribed for 8. The nucleoside 8a was eluated from silica gel column using benzene—ethyl acetate 5:2 (v/v) in 67% yield (2.46 g) Rf = 0.68 (B).

NMR (CDC13): 8.19-7.18 (m., 15H, H-arom.), 5 .75(d . , / = 3.5 H z , H , H / ) , 4 . 7 5 ( s . , 2 H , — OCH?—), 4.07(q,7 = 7 H z , 2 H , - C H 2 - C H 3 ) , 1.12(t . , / = 7 Hz, 3H. - C H 3 ) .

Analysis for C34H30N2OnS (674.6) Calcd C 60.53 H 4.48 N 4.15 S 4.74, Found C 60.72 H 4.21 N 4.32 S 4.95.

7. 5-Methoxycarbonylmethoxy-2-thiouridine (2)

The nucleoside 8a (1.68 g, 2.5 mmol) was dissolv-ed in 0.01 N solution of sodium methanolate in methanol (250 ml) and the reaction was allowed to proceed for 48 h. The solution was neutralized with Dowex 50 W-X-4 (H+form), the resin filtered off and washed with methanol (2x50 ml). The filtrates were combined and methanol evaporated. The residue was extracted with hot diethyl ether (3x50 ml) and crystallized from methanol to give 2 (690 mg, 80%) m.p. 166-167 °C; Rf = 0.58 (B).

UV (H 2 0) : pH = 2, Amax(e) = 282 (8300); pH = 12, Amax(e) = 249 (15,500), 273 (9800).

NMR (CD3OD): 8.17 (s., H, H6), 6.58 (d., J = 1.5 Hz, H, Hi ' ) , 4.69 (s., 2H, - O C H 2 - ) , 3.76 (s., 3H, - C H 3 ) .

MS: mle 348 (0.5%, M+ ) , 256 (0.7%, B+41) (24), 216 (100%, B + H), 157 (87%, B - C O O C H 3 ) .

8. 5-Methoxy-2-thiouridine (1) The nucleoside 8 (4.2 g, 7 mmol) was treated with

0.01 N solution of sodium methanolate in methanol (700 ml) according to procedure described for 8 a to give 1 (1.7 g, 83%) Rf = 0.31 (B) m.p. 229-230 °C (from methanol).

UV (H.O): pH = 2, Amax(e) = 287 (16,500); pH = 12, Amax(e) = 249 (21,800), 273 (13,000).

NMR (CD3SOCD3): 8.36 (s., H, H6), 6.86 (d., J = 2 Hz, H, H, ' ) , 3.95 (s., 3H, - C H 3 ) .

MS: mle 290 (1.7%, M+) , 158 (100%, B + H).

This work was supported by the Ministry of Sci-ences and Education Grant P .R. I I 10.

359 A. J. Malkiewicz-B. Nawrot • The 2-Thioanalogues of rRNA Components

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[19] I. Iwai, S. Nishimura, and B. Shimizu, in W. W. Zür-bach and R. S. Tipson (eds): Synthetic procedures in nucleic acid chemistry, J. Wiley and Sons, New York (1968), Vol. I, p. 388.

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[21] H. Vorbrüggen, in R. T. Walker, E. DeClercq, and F. Ekstein (eds): Nucleosides analogues, chemistry, biology and medical applications. Plenum Press, N.Y. (1979), pp. 35-69 .

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