Glycosylation methods

The first chemical glycosylation was reported in 1879 by Arthur Michael [233]. This reaction proceeded by the nucleophilic displacement of an anomeric leaving group. For this reaction it was necessary to convert the glycosyl acceptor into potassium salt (Figure 34A). A different approach was pursued in 1893 by Emil Fischer, who perceived the unprotected monosaccharide unit as a hemiacetal and the reaction was carried out under harsh acidic conditions, in an excess of the desired glycosyl acceptor (most commonly low weight alcohols that limited the application of this method) (Figure 34B) [234]. In 1901, Koenigs and Knorr (and independently Fischer and Armstrong) presented the most widely used method for the stereospecific synthesis of 1,2-trans glycosides [235]. Glycosylation was obtained by reacting glycosyl halides (bromides and chlorides) as an effective glycosyl donor with conventional alcohol acceptors in

76

the presence of insoluble salts such as Ag2CO3 or Ag2O as well as soluble such as AgOTf and AgClO3 (Figure 34C) [236].

Figure 34 Glycosylation reactions: (A) Michael, (B) Fischer, (C) Koenigs-Knorr and (D) Schmidt. Figure adapted from [236, 237].

The Koenigs–Knorr glycosylation reaction is very selective, often providing complete inversion of the anomeric configuration. A modification of this method according to Helferich employs mixtures of mercury salts such as HgBr and Hg(CN)2 [238]. Further, Ag2CO3, Ag2O, HgO, CdCO3, S-collidine and TMU were frequently used as an acid scavenger and water was generally removed by molecular sieves during these glycosylation reactions. Since the Koenigs–Knorr reaction and its variants requires the usage of often unstable glycosyl halides and heavy metal salt promoters, they have largely been replaced by newer methods [239]. In 1980, Schmidt presented trichloroacetimidates for O-glycosylation reactions. O-Glycosyl trichloroacetimidates are prepared via the addition of trichloroacetonitrile (Cl3CCN) under basic conditions to a free anomeric hydroxyl group using BF3·OEt2 or TMSOTf as promoters (Figure 34D) [237]. Nowadays, most glycosyl donors have non-halogen leaving groups and are typically activated with Lewis acids. Among many leaving groups are fluorides that are not considered with the usual halides, since they are more stable and are activated

77 by different promoters. Widely used donors are glycosyl trichloroacetimidates, thioethers and fluorides. A representative selection of glycosyl donors and activation methods is given in Table 1 [240].

Table 1 Leaving group and appropriate promoters for glycosylation reaction [240].

Leaving group Promoters Leaving group Promoters

F

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3.1. Motivation

In order to understand the role of PARP in many cellular functions (Figure 9) the identification of its interaction partners is extremely important (Figure 7). The key molecule for understanding many processes in the PARP field is PAR, which is synthesized by polymerization of NAD⁺ by PARPs that catalyze the nucleophilic replacement of nicotine amide by the 2'-OH of adenosine. Thus, PAR consists of a nucleotide moiety (adenosine) and a furanose moiety (ribose) that are connected via an 2′-O-adenosine-α-1-ribose glycosidic bond. These units are connected via pyrophosphate linkages (Figure 35). PARG catalyzes the hydrolysis of the mentioned glycosidic bond in endo and exoglycosidic modes of action, thus the intracellular half-life of PAR is less than 1 min (Figure 35C). Since the metabolism of PAR is very dynamic, it is extremely difficult to detect natural PAR. Moreover, knockout organisms that are not possessing PARG are lethal. It is clear that it is highly energy consuming, when the lack of NAD⁺ is occurring because of the fast production of PAR.

Moreover it is not possible for cells to keep long polymers of PAR. Furthermore, the interaction of interaction partners of PARP is difficult due to the presence of PARG in cell extracts, an enzyme that is able to degrade PAR efficiently.

The long term goal of this project is to identify hitherto unknown binding partners of poly(ADP-ribosyl)ated proteins directly from cell extracts. To achieve this goal, new analogues of PAR that mimic the function of PAR but are resistant towards PARG cleavage (Figure 35A) were designed and synthesized.

In order to achieve PARG-resistance without significantly affecting the overall structure and recognition properties of PAR, the development of a synthetic route of PAR analogues, in which one relevant oxygen atom of the glycosidic bond was replaced by an isosteric methylene group resulting in the carbasugar (Figure 35B) target structure, was aimed. . One of the milestones of this project was the synthesis of PAR analogue chains of up to 10 residues that will later be conjugated to proteins. These conjugates in turn will be employed for the identification of hitherto unknown binding partners of PAR. The most challenging, albeit crucial part of the synthesis of the depicted targets is the synthesis of the connection between the nucleoside and sugar part. For the synthesis of the polymer, a corresponding

79 building block is needed (Figure 35B). Since not many modifications of adenosine at the 2′OH position are known, it is necessary to establish an efficient synthesis of the carbasugar with different leaving groups, which will be used for the synthesis of the monomer.

Figure 35 (A) polymer which mimics PAR but it is PARG resistant, (B) carbosugar target structure as PARG-resistant PAR analogues, (C) crucial part of PAR showing PARG action.

Here, the synthesis of carbasugar with different leaving groups is reported.

3.2. Results and discussion 3.2.1. Synthesis of carbasugar

Nucleosides where the furanose moiety is replaced with a cyclopentane or cyclopentene are displaying important antitumor or antiviral activities [241].

Aristeromycin which was first isolated in 1967 and neplanocin A which was isolated in 1981 from Ampullariella regularis are two naturally occurring carbocyclic nucleosides. They were shown to be coproduced at low levels in Streptomyces citricolor [242]. Among the different members of the neplanocin family, neplanocin A has been extensively studied since it showed potent

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antiviral and anti-tumor activity, with significant anti-leukemia activity [243] both in vitro and in vivo. The absence of a true glycosidic bond makes carbocyclic nucleosides chemically more stable and therefore they are not subjected to the action of enzymes that cleave this linkage. A modification of the cyclopentane ring into a cyclopenten ring makes a remarkablely change in the biological activity, leading to increased biological potency and specificity of the unsaturated compounds. This phenomenon was first observed after the isolation and evaluation of neplanocin A, which proved to have superior antitumor and antiviral properties compared with its saturated counterpart aristeromycin (Figure 36) [244, 245]. Since the isolation of neplanocin A, numerous studies have been performed as well as analogues were synthesized [245]. Among others, a fluorocyclopentene analogue, fluoroneplanocin A as well as a cytidine analogue were reported to have antitumor and antiviral activities, e.g 5-fluorocytosine analogue exhibited potent antiviral activity against West-Nile virus [246].

Figure 36 Chemical strucures of Aristeromycin and Neplanocin A.

Due to this reason many different chemical synthesis of neplanocin A as well as of its analogues were reported. However, structure-activity relationship (SAR) studies of carbocyclic nucleosides are hampered by difficulties in preparing the D-carbocycle [247]. Although many methods for the synthesis of the carbocyclic moiety have been reported to date, they suffered from low overall yields, lengthy synthetic routes, racemization, lack of large-scale preparations, and sensitivity to the reaction conditions such as temperature and moisture [247]. Since a total chemical synthesis of one enantiomer, with high yield and relatively less steps to the D-carbocycle is highly desirable, many efforts are currently underway. Ohno and co-workers were the first who

81 published the enantioselective synthesis of aristeromycin and neplanocin A based on the selective hydrolysis of diester (which was constructed via cis hydroxylation of the Diels-Alder adduct) by pig liver esterase [248]. By a series of steps, this intermediate was converted to the cyclopentenylamine, which served as a direct precursor of neplanocin A [248]. The second enantioselective synthesis of neplanocin A, conceptually differs from the already presented by Ohno et al. and was developed by Marquez et al. in 1983 [249]. Altenbach et al.

[250] published in 1985 an identical approach to that by Marquez et al. for the synthesis of cycloalkenones from γ and δ lactone precursors. Next, Johnson and co-workers [251] reported in 1987 the enantioselective synthesis of neplanocin A, which converged with Marquez et al. [245] at the same (1S,4R,5S)-(-)-3-[(Benzyloxy)methyl]-4,5-O-isopropylidene-2-cyclopenten1-ol, which was generated from cyclopentenone. Later on, in 1988, the detailed methodology which was claimed to be simpler and easily adaptable to large-scale syntheses of neplanocin A and other cyclopentene-containing carbocyclic nucleosides were presented [245]. This strategy was based on a chiral carbohydrate precursor to gain control of the three stereochemical centers of the molecule followed by efficient synthesis of cyclopentenone, from either D -ribose or the protected D-ribonic acid lactone, from which the cyclopentenylamine was stereoselectively generated. For the synthesis of carbasugar with different leaving groups, the procedure described by Marquez [245] was followed. Commercially available D-ribono-1,4-lactone was subsequently protected with the isopropylidene and benzyl group (Figure 37).

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Figure 37 Schematic synthesis of (1S,4R,5S)-(1)-3-[(Benzyloxy)methyl]-4,5-O-isopropylidene-2-cyclopenten-1-bromide (a) BnBr, NaH; 0 °C, 16 h, 71 % (b) LiCH2P(O)(OCH3)2; -70 °C, 2 h, 64 % (c) NaOCH3; r.t.; 24 h, 97 % (d) CrO3/pyridine r.t.; 1 h, 64 % (e) K2CO3/18-crown-6; 56 °C, 40 min, 37 % (f) LiAlH4, THF, 0 °C, 15 min, 71 % (g) Ph3P, CBr4, r.t.; 1 h, 51 % [245].

Followed by the treatment of the resulting (-)-5-O-benzyl-2,3-O-isopropylidene-D-ribonolactone (12) with lithium dimethyl methylphosphonate in THF gave the hemiketal 13 in 64 % yield. Subsequent opening of this hemiketal intermediate to the keto phosphonate 14 was accomplished with sodium methoxide in methanol in 97 % yield. Compound 14 exists in the open-chain tautomer of 13, which under acidic conditions can recyclize back to the hemiketal. For this reason, the reaction mixture was carefully neutralized to pH 7 before proceeding with the extraction of 14 with ethyl acetate. Oxidation of 14 using a modified Collins reagent produced the diketo phosphonate 15 (64 % yield), which subsequently underwent intramolecular cyclization to the desired cyclopentenone 16 in not satisfactory 37 % yield. The yield of last step of synthesis compound 16 was low, presumably due to partial base catalyzed epimerization of carbons 3 and 4 in the starting diketone 15, which accounted for an observed partial racemization of the cyclopentenone product.

Unfortunately, enantiomer 16a remained in solution during the crystallization from a mixture of ether and petroleum ether. It was possible to obtain

83 compound 16a with traces of compound 16b with only 6 % overall yield. For this reason, the reduction of compound 16 with lithium aluminium hydride and then subsequent bromination of compound 17 resulted in an overall yield of the synthesis of the carbasugar (18) of only 3 %. Since this was not satisfactory for further applications of carbasugar, a new method of synthesis carbasugar was applied.

One of the new approaches used the 2-cyclopenten-1-one, prepared from (+)-γ-lactone-D-ribonic acid [252-254] was used, followed by a stereoselective reduction to an allylic alcohol. This involves an intramolecular Wittig reaction from a 1,4-diketo derivative prepared from chiral aldonolactones.

Nevertheless, it is well reported that 1,4-chiral diketo derivatives could undergo racemization, under basic media [245]. This of course limits their use as optically pure starting materials [243]. Jacobson and co-workers used olefin ring closing metathesis (RCM) as a key step for the synthesis of carbocycle moieties of neplanocin A from D-ribono-γ-lactone [255].

Next, Jeong et al. [247] and Chu et al. [256] reported short and efficient syntheses of D- and L-3-unsubstituted cyclopentenones, which also employ RCM as the key step and these substrates serve as versatile precursors for the synthesis of D- and L-carbocyclic nucleosides. Another procedure was also reported to obtain enantiomerically pure cyclopentene. The synthetic sequence involves the conversion of the known tetra-O-benzyl-D-galactopyranoside to diene, which is cyclized via RCM reaction [243].

The advantage of the synthesis reported by Jeong is the stereoselective formation of the tertiary β-allylic alcohol 13 from Grignard reaction, which is enforced by the bulky protecting group, and the oxidative rearrangement of the tertiary α-allylic alcohol to the key molecule 15. This synthetic strategy does not work with carbasugar with benzyl as a protecting group because the tertiary chromate ester of such a derivative could not form the six-membered transition state to be rearranged to carbasugar due to the steric hindrance by the 2,3-isopropylidene group. Thus, the next goal was to increase the formation of the tertiary allylic β-alcohol. This was achieved by changing the benzyl protecting group to bulky trityl (Tr) group, which are supported to control the stereoselectivity in the Grignard reaction.

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Figure 38 Schematic synthesis of (4R,5R)-(+)-3-Triphenylmethyloxymethyl-4,5-O-isopropylidene-2-cyclopentenone (a) c-H2SO4, acetone, r.t., 2.5 h, 78 %; (b) TrCl, pyridine, r.t., 48 h, 94 %; (c) Wittig:

methyltriphenylphosphonium bromide in THF, potassium tert-butoxide, 0 °C, 3 h, 90 %; (d) Swern: oxalyl chloride in DCM, DMSO, triethylamine 78 °C, 1 h, 94 %; (e) Grignard: vinylmagnesium bromide in THF, -78 °C, 1h, 96 %; (f) RCM: tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene][benzylidine]ruthenium- (VI) dichloride in methylene chloride, r.t., 48 h, 89 %; (g) PDC: pyridinium dichromate in DMF r.t., 48 h, 73 % [247].

Following this protocol, D-ribose (Figure 38) was protected with isopropylidene and subsequent tritylation and 20 was obtained in 94 % yield.

Followed by Wittig reaction to gave 21 in 90 % yield and Swern oxidation to obtain compound 22 (94 % yield). Grignard reactions of 22 with vinylmagnesium bromide afforded the dienes 23 in 96 % yield. Thus, it is evident that the size of the protecting group plays a major role in controlling the stereochemistry of the carbon carrying the tertiary hydroxyl group during the Grignard reaction. Ring Closing Metathesis (RCM) reaction of 23 using second generation of Grubbs catalyst resulted in the exclusive formation of the desired tertiary β-alcohol 24 in 89 % yield. With the allylic alcohol 24, an oxidation reaction using the PDC in DMF gave the corresponding cyclopentanone 25 (73 % yield), which is the starting material for the synthesis of carbasugar with different leaving groups.

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Figure 39 Schematic synthesis of carbasugar with different leaving groups: (a) CeCl3, NaBH4 (b) LiAlH4, THF -5 °C, 2 h, 78 %; (c) DEAD, PPh3 PhCOOH, NaOH/MeOH, 40 %; (d) MsCl THF Et3N, 60 %; (e) DBU, CCl3CN, (ClC2)2, 88 %; (f) Ph3P, CBr4 51 %.

The cyclopentenone 25 was then reduced with lithium aluminium hydride

to α-alcohol 26 (78 % yield) which was further brominated using:

triphenylphosphine and tetrahalomethanes (CBr4) which is a standardized method known as Apple reaction [257], giving 30 in 53 % yield. The reduction of 25 with sodium borohidride was not successful therefore the convertion of stereocentere under Mitsunobu conditions was performed to obtain 27 in 40 % yield, β-hydroxyl. Carbasugar (27) were also converted to mesyl (28) (60 % yield) and imidate (29) (in 88 % yield) (Figure 39).

3.2.2. Synthesis of artificial monomeric PAR

Another goal of the present thesis was the synthesis of artificial monomeric PAR. Several different conditions were tested; however, all reactions were unsuccessful. In these reactions adenosine, 3,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1.3-diyl) N⁶-(N,N'-dimethylaminomethylene)adenosine

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as well as 3’, 5’-O-(di-tert-butylsilanediyl)- N⁶-(N,N'-dimethylaminomethylene) adenosine were used with following carbsugars: (1)-3-[(benzyloxy)methyl]-4,5-O-isopropylidene-2-cyclopenten-1-ol (17), (1S,4R,5S)-(1)-3-[(benzyloxy)methyl]-4,5-O-isopropylidene-2-cyclopenten-1-bromide (18),

(3aS,4S,6aR)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (26), cyclopenten-1-mesyl (28), (1S,4R,5S)-(1)-3-(trityl)-4,5-O-isopropylidene-2-cyclopenten-1- trichloroacetimidate (29), (1S,4R,5S)-(1)-3-(trityl)-4,5-O-isopropylidene-2-cyclopenten-1-bromide (30).

Carbsugar with a bromide leaving group (18 and 30) was supplied for the reaction with 3,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1.3-diyl) N⁶ -(N,N'-dimethylaminomethylene)adenosine or 3’, 5’-O-(di-tert-butylsilanediyl)- N⁶ -(N,N'-dimethylaminomethylene) adenosine with sodium hydride in different solvents:

DMF and THF, at room temperature as well as in a broad range of temperatures in a microwave 60 - 95 °C. Furthermore, tris[2-(2-methoxyethoxy)ethyl]amine in acetonitril (Seela method) [258], Mitsunobu, silver oxide in following solvents: dichloromethane, THF, toluene; silver tetrafluoroborate with 2,4,6-trimethylpyridine in dichloromethane; and superbase: BEMP in DMF were tested. Following trials to couple unprotected adenosine with carbasugar (30) in the presence of NaH, with and without tetrabutylammonium iodide (TBAI) in DMF at room temperature, as well as in microwave heating from 40 °C up to 160 °C were carried out. Moreover, following conditions to obtain monomer from adenosine and carbasugar (30) were applied: potassium tertbutoxide in DMF, butyl litium in DMF, sodium metoxide in DMF, silver oxide in DMF, silver tetrafluoroborate with 2,4,6-trimethylpyridine in dichloromethane and acetonitrile, superbase: tBuP4 in DMF, sodium hexamethyldisilazide with TBAI in DMF and potassium hydroxide in DMSO. (1S,4R,5S)-(1)-3-(trityl)-4,5-O-isopropylidene-2-cyclopenten-1-mesyl (28) were also employed with unprotected adenosine using the following conditions: sodium hydride in DMF, potassium tertbutoxide in DMF, n-butyl litium in DMF, sodium metoxide in DMF; sodium hexamethyldisilazide with TBAI in DMF. (1S,4R,5S)-(1)-3-(trityl)-4,5-O-isopropylidene-2-cyclopenten-1-

87 trichloroacetimidate (29) was also used with TMSOTf in dichloromethane with protected adenosine.

Furthermore, also trials with isopropanol and bromocarbasugar were performed under the following conditions: potassium tertbutoxide in DMF, silver oxide was used as promotor in acetonitrile, dichloromethane, DMF, THF and toluene as well as silver tetrafluoroborate with 2,4,6-trimethylpyridine in dichloromethane without success.

3.3. Conclusions and outlook

The key molecule (4R,5R)-(+)-3-triphenylmethyloxymethyl-4,5-O-isopropylidene-2-cyclopentenone (15) was successfully synthesized from D -ribose in 7 steps in 25 % overall yield, increasing it 4 times in comparison to the synthesis of cyclopentenone with benzyl protective group (6). This synthetic method can be regarded as an excellent procedure from many different points of view. This total synthesis has seven steps, heigh overall yields, large-scale preparation, and mild reaction conditions.

This compound has a great potential in further reactions for obtaining different carbasugars with different leaving groups, as well as various protection groups. This group of molecules can further be used for establishing methods for the modification of adenosine on 2’OH. To achieve this goal, the carbasugars containing hydroxy (16), bromo (18), mesyl (28) and imidate (29) were synthesised. All of them are promising compounds for the synthesis of monomer of artificial poly(ADP-ribose) PAR.

Different glycosylation methods such as Koenigs and Knorr, Schmidt method as well as different leaving groups were used to synthesise the monomer of artificial PAR. Unfortunately, using these procedures, no product was obtained. Further trials using different protecting groups and conditions are needed to obtain the desired key molecule.

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4. Materials and methods

4.1. General

4.1.1. Chemicals and solvents

All chemicals were - if not stated otherwise - of p.a. or of molecular biology quality grade. Chemicals for synthesis were bought from Acros, Fluka, Sigma Aldrich or Carbosynth and were used without further purification. Dry solvents were purchased from Fluka, solvents for column chromatography were either distilled from technical grade (dichloromethane) or purchased as chromatography grade (hexane, ethyl acetate, methanol, acetonitrile). For chemical synthesis desalted water was used and for experiments involving enzymes, or nucleotide diphosphates water was drawn from a combined reverse osmosis / ultrapure water system (Sartorius, arium 611). Intermixtures of solvents are stated as percent by volume.

4.1.2. General experimental details

All temperatures quoted are uncorrected. All reagents are commercially available and used without further purification. All solvents are dried over molecular sieves and used directly without further purification. All reactions were conducted under exclusion of air and moisture. Purification of monophosphates was performed on a BioLogic DuoFlow System (Bio-Rad Laboratories) with DEAE Sephadex™ A-25 (GEHealthcare Bio-SciencesAB) column using a linear gradient of triethylammonium bicarbonate buffer (TEAB, pH 7.5) (0.1 - 1.0 M, flow 2 mL/min, pH = 7.5). For medium pressure liquid chromatography (MPLC), a Büchi unit with a Büchi controller C-620, two pumps C-605, a UV monitor C-630 (λ = 254 nm) and fraction collector C-660 was used.

NMR spectra: Bruker Avance III 400 MHz spectrometer. ¹H chemical shifts are reported relative to the residual solvent peak and are given in ppm (δ). Flash chromatography: Merck silica gel G60. TLC: Merck precoated plates (silica gel 60 F254). The reported yield refers to the analytically pure substance and is not optimized.

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4.2. Synthesis of NAD

⁺

analogues

2′-O-(propagyl)-adenosine (3, 4)

Adenosine (2 g, 7.48 mmol, 1 eq) were dissolved in 40 mL of hot abs. DMF and cooled to 5 °C, before NaH (0.23 g, 9.73 mmol, 1.3 eq) and propargyl bromide (0.87 mL, 9.73 mmol, 1.3 eq) were added then the reaction mixture was reheated to 55

°C and stirred for 48 h. The solvent was removed under reduced pressure and residue was purified by column – chromatography (3 % methanol in dichloromethane), Rf h. Reaction was quenched with NaHCO₃, extracted with ethyl acetate and washed with a brine, before the organic layer

90

2′-O-(propagyl)adenosine (3)

To a stirred solution of 5 (0.941 g, 1.7 mmol, 1 eq) in 23 mL THF was added tetra-n-butylammonium fluoride (TBAF) (3.4 mL, 3.4 mmol, 2 eq) and stirred at room temperature for 40 min. After evaporation the residue was purified by MPLC (in 3 % methanol in dichloromethane), Rf value: 0.325 obtaining 0.43 g of 3 in 83 % yield. MS-ESI calculated for [M+H]⁺ 506.1, found 506.5.

1H NMR (400 MHz, Methanol-d4) δ 8.33 (s, 1H, H-2), 8.21 (s, 1H, H-8), 6.10 (d, J = 6.5 Hz, 1H, H-1′), 4.87 (dd, J = 6,4, 5,0 Hz, 1H, H-2′), 4.53 (dd, J = 4.9, 2.5 Hz, 1H, H-3′), 4.30 (dd, J = 16.1, 2.4 Hz, 1H, CH2), 4.25 (dd, J = 16.1, 2.4 Hz, 1H, CH2), 4.21 (q, J = 2.5 Hz, 1H, H-4′), 3,91 (dd, J = 12,6, 2,4 Hz, 1H, H-5′a), 3,77 (dd, J = 12,6, 2,6 Hz, 1H, H-5′b), 2.64 (t, J = 2.4 Hz, 1H, CH).

13C NMR (101 MHz, MeOD) δ 157.59 (C-6), 153.54 (C-4), 150.12 (C-2), 142.05 (C-8), 120.99 (C-5), 89.33 (C-1′), 81.71 (C-4′), 79.82 (C-2′), 76.54 (propagyl C), 71.16 (propagyl =CH), 63.44 (C-3′), 59.58 (C-5′), 58.78 (propagyl OCH₂).

2′-O-(propagyl)adenosine monophosphate (6)

The 2′-O-(propargyl)adenosine 3 (100 mg, 0.327 mmol, 1eq) was dried over night with proton sponge (105 mg, 0.49 mmol, 1.5 eq) under reduced pressure. The dried starting material was dissolved in 3 mL of P(OMe)₃, cooled to -20 °C and added 80 µL distilled POCl₃. Reaction control through TLC (isopropanol/water/NH₂OH = 6/1/1) after stirring for 4 h showed still starting material, so 80 µL of POCl₃ were added

again. TLC after further 30 min showed arising side products. The reaction was stopped through addition of the 0.1 M TEAB buffer solution and purified with FPLC obtaining 2′-O-(propargyl)adenosine monophosphate (6) (92 mg, 0.241 mmol) in 73 % yield. MS-ESI calculated for [M+H]⁺ 385.0, found 385.9.

1H NMR (400 MHz, Deuterium Oxide) δ 8.45 (s, 1H, H-2), 8.17 (s, 1H, H-8), 6.14 (d, J

= 6.4 Hz, 1H, H-1′), 4.71 (dd, J = 6.4, 5.2 Hz, 1H, H-2′), 4.59 (dd, J = 5.2, 3.0 Hz, 1H, H-3′), 4.36 – 4.32 (m, 1H, H-4′), 4.28 (dd, J = 16.2, 2.4 Hz, 1H, CH₂), 4.23 (dd, J = 16.2,

91 mmol, 0.01g) was dissolved in a mixture of water (0.25 mL) t-butanol (0.25 mL) and morpholine (10.4 µL), the reaction mixture was refluxed and DCC (0.026 g) was added slowly in t-butanol (0.37mL). Reaction was refluxed for 2 h and then

cooled to room temperature. Dicyclohexyl urea was filtered. Reaction was purified with MPLC 50 mM TEAB buffer with acetonitril giving compound in 27 % yield (0.006 mmol, 3 mg). MS-ESI calculated for [M+H]⁺ 455.4, found 455.9.

1H NMR (400 MHz, Deuterium Oxide) δ 8.45 (s, 1H, H-2), 8.21 (s, 1H, H-8), 6.17 (d, J mmol)in glove box. To dried overnight mixture of adenosine monophosphate imidazolidate (3 mg, 6.62 *10^-3 mmol),

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μL) in formamide. The resulting suspension was stirred at room temperature for 48 h.

μL) in formamide. The resulting suspension was stirred at room temperature for 48 h.

Im Dokument "Clickable" chain terminator NAD⁺ analogs for labeling substrate proteins of poly(ADP-ribose) polymerases (Seite 75-0)