First total synthesis of kipukasin A

In this paper, a practical approach for the total synthesis of kipukasin A is presented with 22% overall yield by using tetra-O-acetyl-β-D-ribose as starting material. An improved iodine-promoted acetonide-forming reaction was developed to access 1,2-O-isopropylidene-α-D-ribofuranose. For the first time, ortho-alkynylbenzoate was used as protecting group for the 5-hydoxy group. After subsequent Vorbrüggen glycosylation, the protecting group could be removed smoothly in the presence of 5 mol % Ph3PAuOTf in dichloromethane to provide kipukasin A in high yield and regioselectivity.


Introduction
Endogenous nucleosides are involved in DNA and RNA synthesis, cell signalling, enzyme regulation and metabolism etc. [1,2]. Therefore, the synthesis of novel nucleosides to mimic their physiological counterparts has potential therapeutic significance, which has led to the development of a large number of antiviral and antitumor drugs [3,4]. On the other hand, naturally occurring nucleosides, especially marine nucleosides, have also played an indispensable role in drug discovery, which make great contribution in the commercialization of cytosine arabinoside (Ara-C), adenine arabinoside (Ara-A) and AZT, etc. [5,6]. Nucleosides and their analogues will continue to play an important role in future drug discovery [7].
Some of them showed promising antibiotic, antiviral, antiparasitic and antitumor properties. Kipukasins A-G were firstly isolated from solid-substrate fermentation cultures of Hawaiian Aspergillus Versicolor in 2007 ( Figure 1) [11]. Later on, kipukasins H, I [12] and J [13] were also isolated from the fungus Aspergillus flavus, which was collected at the South China Sea and the Sea of Okhotsk, respectively.
Kipukasins are uridine derivatives with unique structural characteristics, which include: (1) a uracil moiety with or without an N-3 methyl group; (2) a 6-methyl-2,4-hydroxy (or methoxy)benzoyl group at C-2' or C-3' position; (3) with or without an acetyl group at 2'-OH position. To the best of our knowledge, they are the first naturally occurring aroyl nucleosides reported up to now. The biological assays showed that kipukasin A  owned modest activity against Gram-positive bacteria Staphylococcus aureus (ATCC 29213) [11].
During our ongoing biological studies of marine nucleosides, total syntheses of several marine nucleosides were accomplished in our group [14][15][16][17][18]. In the present paper, we reported a practical approach for the total synthesis of kipukasin A.
to occur in nucleosides [19][20][21]. The synthetic route would be lengthy and cumbersome. Therefore, a practical total synthesis is in high demand to facilitate the preparation of other kipukasins and their analogues.
The retrosynthetic analysis is shown in Figure 2 (path b). Kipukasin A could be constructed by Vorbrüggen glycosylation [22,23] of a properly protected glycosyl donor 3 with uracil (4). Neighboring group participation of the 2'-O-acetyl group stereoselectively facilitate the β-glycosidic bond formation. Thus, the choice of a suitable protecting group at 5-OH position would be crucial for the success. It should fulfill at least two requirements: (1) it should be stable during the Vorbrüggen glycosylation; and (2) the deprotection process should be performed under very mild and neutral conditions without any influence on the 2'-O-acetyl group. At the same time, ester protection is preferred for Vorbrüggen glycosylations in nucleoside syntheses. Very recently, ortho-alkynylbenzoate was successfully developed by our group as neighboring participation group to synthesize 2'-modified nucleosides [24], which could be removed smoothly in the presence of gold(I) complexes with high yield and selectivity. The conditions are very mild and neutral. In the present paper, we continue to use ortho-alkynylbenzoate as protecting group for the 5'-OH group to fulfill the total synthesis of kipukasin A.
Subsequently, using DMAP as acylation catalyst and triethylamine as base, the former synthesized 2,4-dimethoxy-6-methylbenzoyl chloride (9) reacted with ribose 14 to 3-O-(2,4dimethoxy-6-methylbenzoyl)ribose 15 in 74% yield. Various spectral analyses (NMR, HPLC) showed no evidence that 2,3-O-transesterification occurred during the esterification reaction. After cleavage of the acetonide group with acetic acid/acetic anhydride/H 2 SO 4 , the key glycosylation donor 16 was obtained in 74% yield as a mixture of isomers (α/β = 1:8) [36].  With glycosylation donor 16 in hand, we proceeded to investigate the crucial Vorbrüggen glycosylation with uracil (4). To our delight, in a similar manner as our described in [17], it proved to be efficient to give nucleoside 17 with exclusive β-configuration in 89% yield. At last, using our developed approach [24], kipukasin A was obtained in 90% yield in the presence of 5 mol % Ph 3 PAuOTf in dichloromethane with H 2 O (1 equiv) and ethanol (6 equiv). All spectra of the synthetic kipukasin A were consistent with an authentic sample.

Conclusion
In summary, the first total synthesis of kipukasin A was accomplished with 22% overall yield. The reaction sequence includes: (1) an improved iodine-promoted acetonide-forming reaction to synthesize 1,2-O-isopropylidene-D-ribofuranose (12); (2) a Vorbrüggen glycosylation facilitating the preparation for kipukasin derivatives and (3) the first use of ortho-alkynylbenzoate as protecting group of the 5-hydoxy group, which can be removed smoothly in the presence of 5 mol % Ph 3 PAuOTf in dichloromethane. Biological studies of kipukasin A and the total synthesis of other kipukasin nucleosides by this established approach are ongoing in our group.

Experimental
All reagents and catalysts were purchased from commercial sources (Acros or Aldrich) and used without purification. DCM and CH 3 CN were dried over CaH 2 and distilled prior to use. Et 3 N was dried over NaH and distilled prior to use. Thin-layer chromatography was performed using silica gel GF-254 plates with detection by UV (254 nm) or charting with 10% sulfuric acid in ethanol. Column chromatography was performed on silica gel (200-300 mesh, Qing-Dao Chemical Company, China). NMR spectra were recorded on a Bruker AV400 spectrometer, and chemical shifts (δ) are reported in ppm. 1 H NMR and 13 C NMR spectra were calibrated with TMS as internal standard, and coupling constants (J) are reported in Hz. The ESI-HRMS were obtained on a AB SCIEX Triple TOF 4600 spectrometer in positive ion mode. Melting points were measured on an electrothermal apparatus and are uncorrected.
Optical rotation values were measured with a Rudolphautopol IV polarimeter.

Synthesis of 1,2-O-isopropylidene-α-D-ribofuranose (12):
The light-yellow oil 11 (7.60 g, 27.7 mmol) was dissolved in MeOH (60 mL). To the solution K 2 CO 3 (0.60 g, 4.4 mmol) was added and the reaction mixture was stirred for 2 h at room temperature. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column to give 12 as a white solid (4.60 g, 93%). R f 0.30 (CH 2 Cl 2 /CH 3

Synthesis of kipukasin A:
To a solution of nucleoside 17 (0.60 g, 0.92 mmol) in dry CH 2 Cl 2 (15 mL) was added H 2 O (1.0 equiv), and ethanol (6.0 equiv) under an argon atmosphere. The mixture was stirred at room temperature for 20 minutes. A freshly prepared solution of Ph 3 PAuOTf in CH 2 Cl 2 (5 mol % in 1.0 mL) was added, and stirring was continued at room temperature for 5 hours until nucleoside 17 was consumed as monitored by TLC. The reaction mixture was filtered with celite. After filtration, the filtrate was evaporated to dryness under reduced pressure. The obtained residue was recrystallized in petroleum ether (5 mL) to provide kipukasin A as a white powder solid (387 mg, 90%). R f 0.36 (CH 2 Cl 2 /CH 3

Supporting Information
Supporting Information File 1 Experimental procedures of compounds 6-9, copies of 1 H and 13 C NMR spectra of all compounds and X-ray crystal data of compound 13.