Julen Ariztia

Alicia Chateau

Cédric Boura

Claude Didierjean

Sandrine Lamandé-Langle

Nadia Pellegrini Moïse
email: nadia.pellegrini@univ-lorraine.fr

Synthesis of anti-proliferative [3.3.0]furofuranone derivatives by lactonization and functionalization of C-glycosyl compounds

Keywords: C-glycosyl compound, furofuranone, anti-proliferative compound

furofuranone structure is found in numerous families of biologically active natural products. We took advantage of the stereodiversity afforded by carbohydrate derivatives to prepare several compounds structurally similar to goniofufurone and crassalactones which are natural cytotoxic agents. We designed and synthesized several stereoisomers of these natural compounds via lactonization of C-glycosyl compounds bearing an hydroxyl on position 4 and a methyl ester on the pseudo-anomeric positionThe reactivity of this bicyclic moiety was explored through etherification of hydroxyls in position 5 and 7 and various substituants (halogen, phenyl, benzyl, cynanmoyl) were introduced. The anti-proliferative properties of these mimics were then evaluated on various cancer cell lines and two compounds 24 and 35 demonstrated IC 50 value of 1.34 µM (U251) and 7.60 µM (U87) respectively.

Introduction

Carbohydrate chemistry is a field of major interest for synthetic chemists because these types of compounds play important roles in biological processes. Moreover sugars are often used as starting chiral materials in total synthesis and are useful scaffolds for the construction of bioactive compounds. Indeed, their polyfunctional structure is really attractive to design bioactive compounds by appending substituents at selected positions which requires the development of efficient synthetic strategies. Close analogs of O-glycosyl derivatives, C-glycosyl compounds display chemical and biological stabilities toward enzymatic hydrolysis and are thus extensively studied. [START_REF] Beau | Glycoscience: Chemistry and Chemical Biology I-III[END_REF][START_REF] Skrydstrup | Carbohydrates in Chemistry and Biology[END_REF][START_REF] Xu | [END_REF][4] In this context, our lab was early involved in the development of C-glycosidic derivatives [START_REF] Pellegrini Moïse | Chapter 6[END_REF][START_REF] Petry | [END_REF][7][8] to build bioactive compounds, [9,10] peptidomimetic derivatives [11] suitable for imaging applications [12] and also for the design and synthesis of foldamers. [13,14] In the course of the synthesis of glycopeptides based on sugar γ-amino acids, we reported the spontaneous formation of a C-glycosyl bicyclic lactone in which the lactone ring is annulated to a furanoid system. [13] The [3.3.0]furofuranone scaffold obtained this way is found in numerous families of biologically active natural and synthetic products explaining the continual interest nowadays in its chemistry. [15][16][17][18][19] Figure 1a shows some natural molecules with [3.3.0]furofuranone structure. (+)-Goniofufurone was isolated from Goniothalamus and the absolute configuration of natural (+)-Goniofufurone was attributed by Shing et al.. [20] Crassalactones B and C are cinnamoyl esters of Goniofufurone isolated from Polyalthiacrassa [21] and their absolute configurations were determined thanks to chemical modification of (+)-Goniofufurone. These natural products and number of their analogues have been the subject of many syntheses. Just to mention some of them, the total synthesis of natural (+)-Goniofufurone was early reported by a non-classical Wittig reaction on furanolactone obtained from D-glucose. [22,23] A great number of total syntheses of (+)-Goniofufurone have been reported [18,[24][25][26][27][28] and a photochemical approach has been recently reported. [29] Crassalactone C is also the subject of many investigations and its first total synthesis was reported from D-xylose in a multistep sequence by Popsavin et al. [30] An expeditious enantiospecific synthesis involving a ring-closing metathesis reaction was also reported by Prasad et al. [31] Together with the development of synthetic approaches, Popsavin's group is fully implicated in the synthesis of many series of lactone derivatives with significant cytotoxic activity against human tumor cell lines. [32][33][34][35][36]. Just to mention few of them, the C7-dephenylated, 7-halogeno and 7-azido-7-deoxy mimics were prepared and showed a potent and selective in vitro cytotoxicity against specific human tumor cell lines. [37][38][39] Conformationally constrained goniofufurone mimics with 1,3-dioxane heterocyclic ring embedded in 5,7-positions were designed and evaluated [40,41] and a series of conformationally restricted analogs with halogen, azido or benzyloxy groups at the C-7 position were recently described. [42] Mimics bearing a methoxy group at C-5 and C-7 positions [43] and C-7-alkyloxymethyl groups (C6-C13) were recently investigated. [44] Related to Crassalactones B and C, Popsavin et al. also designed and evaluated structural features through a 3D-QSAR model for various styryl lactones and the crucial effect of the cinnamic acid ester moiety at C-7 was highlighted. [45,46] Based on these data and considering our expertise in carbohydrates and more particularly in Cglycosyl compounds chemistry, we planned to design and prepare several compounds featuring a furofuranone moiety. Amongst the stereodiversity afforded by carbohydrates, ribo-and gulofuranose derivatives were chosen as starting compounds since they give access to C-5 and C-5/C-6 stereoisomers of (+)-Goniofufurone (Figure 1b). To the best of our knowledge, these stereoisomers featuring a clear structural similarity with goniofufurone and crassalactones have never been The objective of this work was the synthesis of the targeted lactone moiety based on two furanoses and the insertion of various substituents (halogen, phenyl, benzyl, cynanmoyl) selected according to biological evaluations found in the literature. The introduction of these substituents led us to explore the scope and limitation of etherification of this lactone. The anti-proliferative properties of the targeted analogs were evaluated on various cancer cell lines.

Results and Discussion

Chemistry

Our approach for the preparation of the bicyclic furofuranone moiety is based on the intramolecular lactonization of a C-glycosyl compound. Indeed, we previouly described a spontaneous concommitant acetal deprotection and lactonization during acid catalyzed removal of isopropylidene leading to a C-glycosyl bicyclic lactone in which the lactone ring is annulated to a furanoid system. [13] Among the numerous synthetic methods to prepare C-glycosyl derivatives, we developped and used for several years the double bond reduction of C-glycosylidene compounds commonly called exoglycals (compound 2). Thus, starting from commercially available D-ribonolactone 1 (Scheme 1) and D-gulonolactone 18 (Scheme 4), we managed to prepare C-5 and C-5/C-6 stereoisomers of the bicyclic furofuranone moiety of (+)-Gogniofufurone respectively. In line with the most active compounds recently described by Popsavin's group, we selected halogens, phenyl, benzyl and cinnamoyl groups to be introduced at positions 5 or 7.

The C-glycosyl compound 3 was obtained by a three steps synthetic sequence involving hydroxyl protection, Wittig reaction and stereoselective hydrogenation, a classical method used in our laboratory (Scheme 1). [47,48] O In order to prepare mimics bearing a benzyl or phenyl group at C-5 and C-7 positions, the reactivity of the corresponding hydroxyl groups was explored. The protection of the primary hydroxyl group on position 7 was first required (Scheme 2). We planned to adopt a para-methoxybenzyl as protecting group. The reaction with PMB-Cl in presence of NaH leads to the PMB-ester resulting from a transesterification of the methyl ester together with the protection in position 7. This issue was easly circumvented by the use of PMB-trichloroacetimidate with BF 3 .Et 2 O, leading to the 7-O-PMB derivative 9.

We next attempted the concommitant acetal deprotection and lactonisation in acidic conditions. The resulting furofuranone 13 was derivatized in order to prepare a benzylated compound close to the most active one described by Popsavin's group. This prompted us to explore the reactivity of lactone 13 towards etherification, which proved to be challenging. As we suspected that the use of a base could be troublesome given the presence of the lactone moiety, we carefully explored the scope and limitation of this reaction with various commonly used electrophiles and bases (Table 1). Firstly, the reaction was performed with iodomethane and NaH in DMF. The complete degradation of 13 was observed whether the reaction was conducted at 0 °C or -20 °C (entries 1 and 2). The use of dimethyl sulfate instead of iodomethane did not permit to obtain the targeted methylether and interestingly, no reaction occurred and starting compound 13 was totally recovered when sodium carbonate was used instead of sodium hydride (entry 3), the same result was observed with benzylbromide (entry 4). The reaction conducted at 70 °C did not allow the formation of the benzylated compound and led to complete degradation of compound 13 (entry 5). The reaction with benzyl bromide and KHMDS in THF at -80 °C was also unsuccessful (entry 6). We then turned to silver oxyde used for O-alkylation of the primary hydroxyl in the preparation of goniofufurone mimics bearing an alkoxymethyl chain. [44] For O-alkyation of 13, the use of silver oxyde in dichloromethane has proven to be efficicient in a sealed tube at 60 °C during 24 h. In these conditions (entry 7), the attempted 5-O-Bn 15 was obtained in 48 % yield. A prolonged reaction time (48 h) afforded 5-O-Me 14 and 5-O-Bn 15 in high yields (entries 8 and 10). 5-O-allyl 16 was obtained with lower yield but the unreacted starting compound 13 was easily recovered (entry 9). These results clearly showed that the O-alkylation of the lactone 13 is challenging in basic media required for this type of reaction. 5-O-Bn 15 was then treated with PTSA in methanol and afforded compound 17 in 61 % yield. We also prepared C-7-phenylated mimics by nucleophilic addition of phenyl magnesium bromide on aldehyde 21 (Scheme 4). [49] Two C-7 diastereoisomers 29 and 30 were obtained in 65/35 ratio determined by 1 H NMR on the basis of signal integration of H-6 and methyl ester, but these compounds were not separable by column chromatography. In contrast, the polarities of diols 31 and 32 obtained after lactonisation were sufficiently different to allow their separation by column chromatography. X-ray analysis on 32 (Figure 2) umambiguously confirmed that 32 is a C-5,C-6,C-7 stereoisomer and compound 31 is a C-5,C-6 stereoisomer of (+)-Goniofufurone. Finally, a Crassalactone C mimic was prepared by a cinnamoylation of the diasteroisomeric mixture of 29/30.

We were not able to separate the mixture of cinnamoylated diastereoisomers 33/34 but fortunately, the pure major stereoisomer 35 was obtained after lactonization and careful purification by column chromatography.

Biological evaluation and discussion

The is not yet understood, but could be exploited to elaborate more selective chemotherapies. This could prevent several side effects observed in in conventional chemotherapies, such as cardiotoxicity of doxorubicine.

Conclusion

Several compounds featuring a clear structural similarity with goniofufurone and crassalactones which are natural cytotoxic agents were prepared based on the stereodiversity afforded by carbohydrate derivatives. The targeted bicyclic lactone moieties were obtained by intramolecular lactonization between the hydroxyl and a methylcarboxylate of C-glycosyl compounds. The reactivity of the hydroxyl in position 5 was explored and the C-5 and/or C-5/C-6 stereoisomers were etherified with various substituants (phenyl, allyl or methyl) in presence of Ag 2 O. The reactivity of the hydroxyl in position 7 was explored in order to obtain the halogenated, benzylated and cinnamoylated C-5 and/or C-5/C-6 stereoisiomers.Their anti-proliferative properties were then evaluated on various cancer cell lines. Some mimics showed no significant growth inhibitory activity against all tumour cell lines, highlighting that the stereochemistry of the furofuranone moiety is a crucial point to ensure cellular activity. Interestingly, compounds 24 and 35 demonstrated IC 50 values of 1.34 µM on U251 and 7.60 µM on U87 cells, respectively. The cell cycle phases were examined but these experiments did not permit to establish that the cytotoxic effect is due to apoptosis. Nevertheless, the cytotoxic effects observed for these compounds towards distinctive cell lines could be explored in the future in order to consider more specific chemotherapies.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge: Copy of NMR spectra of new compounds and biological assays.

Experimental Part

General information

Solvents and liquid reagents were purified and dried according to recommended procedures. Thin layer chromatography (TLC) analyses were performed using standard procedures on Kieselgel

3,6-anhydro-2-deoxy-4,5-O-(1-methylethylidene)-7-O-(p-methoxybenzyl)-D-ribo-heptanoic acid methyl ester 9

To a solution of 4 (100 mg, 0.40 mmol) in dry dichloromethane (5 mL), 225 mg of 4-methoxybenzyl 2,2,2-trichloroacetimidate (2 eq., 0.80 mmol) were added under inert atmosphere. The reaction mixture was cooled at 0 ºC and 2 µL of BF 3 .OEt 2 (0.04 eq, 0.016 mmol) were added and the mixture was stirred overnight a room temperature. After 16 h, the catalyst was filtered off through a Celite ® pad and the solvent was removed under vacuum.

3,6-anhydro-2-deoxy-4,5-O-(1-methylethylidene)-7-O-(tert-butyldiphenylsilyl)-D-ribo-heptanoic acid methyl ester 11

To a solution of 4 (1 g, 4.05 mmol) in dry DMF (10 mL), a solution of 1.58 mL of tertbutyldiphenylsilyl chloride (1.5 eq., 6.07 mmol) and 496 mg of imidazole (1.8 eq., 7.29 mmol) in dry DMF (3 mL) was added dropwise at 0 ºC under inert atmosphere. The reaction was stirred overnight at room temperature. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3x80 mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum. The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 90/10) to afford compound 11 ( [M+Na] + : 197.0420, found: 197.0401.

3,6-anhydro-2-deoxy-5-hydroxy-7-O-(tert-butyldimethylsilyl)-D-ribo-1,4-lactone 13

To a solution of 12 (140 mg, 0.80 mmol) in dry DMF (4 mL), a solution of 225 µL of tertbutyldiphenylsilyl chloride (1.1 eq., 0.86 mmol) and 120 mg of imidazole (2.2 eq., 1.76 mmol) in dry DMF (2 mL) was added dropwise at 0 ºC under inert atmosphere. The reaction was stirred for 24 h at 40 ºC. The reaction mixture was diluted with water (15 mL) and extracted with EtOAc (3x20 mL).

The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum. The

3,6-anhydro-2-deoxy-5-O-(methyl)-7-O-(tert-butyldiphenylsilyl)-D-ribo-1,4-lactone 14

To a solution of 13 (100 mg, 0.24 mmol) in dry dichloromethane (5 mL), 185 mg of Ag 2 O (3 eq., 0.73 mmol) and methyl iodide (4 eq., 0.96 mmol) were added under inert atmosphere. The reaction was stirred for 48 h at 60 ºC in a sealed tube. The reaction mixture was filtered off through a Celite ® pad and the solvent was removed under vacuum.

3,6-anhydro-2-deoxy-5-O-(benzyl)-7-O-(tert-butyldiphenylsilyl)-D-ribo-1,4-lactone 15

To a solution of 13 (100 mg, 0.24 mmol) in dry dichloromethane (5 mL), 185 mg of Ag 2 O (3 eq., 0.73 mmol) and benzyl bromide (4 eq., 0.96 mmol) were added under inert atmosphere. The reaction was stirred for 48 h at 60 ºC in a sealed tube. The reaction mixture was filtered off through a Celite ® pad and the solvent was removed under vacuum. The crude product was purified by flash

3,6-anhydro-2-deoxy-5-O-(allyl)-7-O-(tert-butyldiphenylsilyl)-D-ribo-1,4-lactone 16

To a solution of 13 (100 mg, 0.24 mmol) in dry dichloromethane (5 mL), 185 mg of Ag 2 O (3 eq., 0.73 mmol) and allyl bromide (4 eq., 0.96 mmol) were added under inert atmosphere. The reaction was stirred for 48 h at 60 ºC in a sealed tube. The reaction mixture was filtered off through a Celite ® pad and the solvent was removed under vacuum. The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford 16 (38 mg).

Yield: 35 %; colorless oil; R f = 0.63 (cyclohexane/EtOAc 6/4); [α] D = 65.1 (c = 0.92, CHCl 3 ); IR (film): ν 2928, 2857 1782, 1471, 1458, 1427 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Sitert-butyl), 2.70 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 2.0 Hz, H-2a), 2.77 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b),

3.75 (dd, 1H, J 7a,7b = 11.5 Hz, J 7a,6 = 3.0 Hz, H-7a), 3.91 (dd, 1H, J 7b,6 = 2.5 Hz, H-7b), 4.01 (app dt, 1H, J 6,5 = 7.0 Hz, H-6), 4.06 (app ddt, 1H, J 8a,8b = 12.5 Hz, J 8a,9 = 6.0 Hz, J 8a,10a = J 8a,10b = 1.0 Hz, H-8a), 4.21 (app ddt, 1H, J 8b,9 = 5.5 Hz, J 8b,10a = J 8b,10b = 1.

3,6-anhydro-2-deoxy-5-O-(benzyl)-7-hydroxy-D-ribo-1,4-lactone 17

To a solution of the benzylated lactone 15 (30 mg, 0.06 mmol) in methanol (2 mL), 23 mg of PTSA monohydrate (2.0 eq., 0.12 mmol) were added. After stirring at 30 ºC (TLC monitoring) the solvent was removed in vacuum and the crude product was purified by chromatography on silica gel (eluent:

3,6-anhydro-2-deoxy-4,5-O-(1-methylethylidene)-7-iodo-D-galacto-heptanoic acid methyl ester

To a solution of 22 (370 mg, 1.50 mmol) in dry THF (3.5 mL), 215 mg of imidazole (2.1 eq., 3.15 mmol) and 826 mg of PPh 3 (2.1 eq., 3.15 mmol) were added. The mixture was heated at reflux and a solution of I 2 (480 mg, 1.90 mmol, 1.25 eq.) solubilized in dry THF was added (1.5 mL). After stirring at reflux until completion of the reaction, the solvent was removed in vacuum and the crude residue was purified by column chromatography (eluent: cyclohexane/EtOAc 100/0 to 20 To a solution of 29/30 (360 mg, 1.12 mmol) in CH 2 Cl 2 (15 mL), 183 mg of trans-cinnamic acid (1.1 eq., 1,23 mmol), 164 mg of DMAP (1.2 eq., 1.34 mmol) and 277 mg of DCC (1.2 eq., 1.34 mmol)

were added. The solution was stirred for 48 h at 25 ºC. The solution was washed with an aqueous solution of 1 M HCl (2x10 mL), a saturated solution of NaHCO 3 (2x10 mL) and brine (10 mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum.

1 HFigure 1 .

 11 Figure 1. a) Natural compounds with the [3.3.0]furofuranone structure. b) Structures of the envisioned stereoisomers. The representation is chosen in order to clearly visualise C-5 and C-6 configurations.

  prepared and could potentially display cytotoxic activity. Most of the time, the synthetic pathway for (+)-Goniofufurone synthesis involves 1,2-O-isopropylidene-D-glucose or 1,2-O-isopropylidene-Dxylofuranose as starting chiral precursors and the most active derivatives described by Popsavin's group are often prepared by O-alkylation prior to lactonization. We propose herein the synthesis of mimics based on the lactonization of a C-glycosyl compound involving the hydroxyl on position 4 (carbohydrate numbering) and a methyl ester on the pseudo-anomeric position.

Scheme 1 .

 1 Scheme 1. Reagents and conditions. a) H 2 , Pd/C, EtOAc, 90 %; b) Na(0), MeOH, 80 %; c: R = I, I 2 , imidazole, PPh 3 ,THF, reflux, 3 h, 77 %; R = F, DAST, CH 2 Cl 2 /ACN, 0 °C-rt, 2 h, 76 %; d: TFA, CH 2 Cl 2 , 0 °C, 3 h, 7: 67 %, 8: 93 %. In the first instance, we envisioned the preparation of 7-halogeno derivatives. The C-glycosyl compound 4 obtained by a Zemplen reaction on compound 3 was reacted with iodide and PPh 3 for iodination and diethylaminosulfur trifluoride (DAST) for fluorination. The targeted 7-iodo and 7fluoro derivatives 7 and 8 were obtained in 67 and 93 % yields respectively after lactonisation during acid catalyzed removal of isopropylidene (Scheme 1).

Scheme 3 .

 3 Scheme 3. Reagents and conditions: a) HCl (1N), MeOH, 3 h, 90 %; b) NaIO 4 , MeOH,91 %; c) NaBH 4 , MeOH, 3 h, 64 %; d) I 2 , imidazole, PPh 3 , THF, 61 %; e) TFA/H 2 O (1/1 v/v), 3 h, 44 % for 24, 62 % for 25; f) TBDPSCl; imidazole, DMF, 18 h, 57 %; g) Ag 2 O, BnBr, CH 2 Cl 2 , 60 ºC, 48 h, 29 %; h) PTSA, MeOH, 30 °C, 18 h, 49 %.

Scheme 4 .

 4 Scheme 4. Reagents and conditions: a) PhMgBr, Et 2 O, 0 °C, ratio 29/30: 65/35; global yield 35 %; b) TFA/H 2 O (1/1 v/v), 31: 25 % and 32: 15 %. c) cinnamoyl carboxylic acid, DCC, DMAP, CH 2 Cl 2 , 53 %, ratio 33/34: 65/35; d) TFA/CH 2 Cl 2 (1/1 v/v), 56 %.

Figure 2 .

 2 Figure 2. Crystal structure of 32, showing 50% probability thermal ellipsoids. Some hydrogen atoms are omitted for clarity.

7 -halogeno compounds 7 , 8 Figure 3 .

 7783 Figure 3. Structure of tested compoundsCytotoxic activity was evaluated using the standard MTT assay after 72 h exposure of cells to the compounds at different concentrations (0.1, 1, 10, 100, 250µM). The commercial antitumour agent Doxorubicin (Dox) was used as reference. As a representative result, the cytotoxic activity of all compounds on U87 cells line is depicted in Figure4and other cancer cell lines results can be found in the SI. The compounds showed weak growth inhibitory activity against all tumour cell lines, except compounds 35 and 24 for which IC 50 values were calculated (Table2). Remarkably, the styryl lactone 35 exhibited toxicity toward U87 and U251 cell lines with IC 50 of 32.4 µM and 1.34 µM respectively. This compound exhibited a lesser effect against MDA-MB-231 and DAOY cells. The iodo derivative 24 demonstrated a significant cytotoxic activity on U87 cells (IC 50 of 7.6 µM). The stereoisomers of goniofufurone 31 and 32 showed no cytotoxic activity. This result revealed that these derivatives did not have the appropriate structural spatial arrangement of the furofuranone segments although they display a clear structural similarity with goniofufurone. The dephenylated analogs 12 and 25, which are respectively C-5 and C-5/C-6 stereoisomers also demonstrated a total loss of activity, although dephenylated derivatives described by Popsavin's group are cytotoxic compounds towards a large panel of cancer cell lines. In a similar way, 5-O-Bn substituted mimics 17 and 28 did not display a significant biological activity. 7-iodo mimics 24 and 7, which are C-6 stereoisomers, displayed completely different results. Based on a D-gulo-configurated scaffold, the 7-iodinated compound 24 and the crassalactone mimic 35 have the highest potency with IC 50 <10 μM on U87 and U251 cell lines.

Figure 4 .

 4 Figure 4. Cytotoxic activity of mimics on U87 cell lines a). Cytotoxic activity on U87, U251, DAOY and MDA-MB-231 cells of compound 35 b), compound 24 c) and Doxorubicin d).

Figure 5 .

 5 Figure 5. Cell cycle of U251 cells treated (72 h) with compounds 24 and 35 (1 µM) and Doxorubicine (0.1 µM)

-heptanoic acid methyl ester 6 To a solution of 4 ( 7 To a solution of 4 ( 5 [ 8 Product 6 (

 6474586 60F254 plates (Merck, Kenilworth, NJ, USA). Compounds were visualized using UV light (254 nm) and a methanolic solution of sulfuric acid and charred. Column chromatography was performed on silica gel SI 60 (63-200 μm) (Merck). Melting points were determined with a Tottoli apparatus and are uncorrected. FTIR spectra were recorded on a Shimadzu IRAffinity-1, ATR PIKE Technologies model GladiAT (cm-1). Optical rotations were measured on an Anton-Paar MCP 300 polarimeter.1 H and13 C NMR spectra were recorded on a Bruker Avance III 400 (400 MHz and 100.6 MHz, respectively). For complete assignment of 1 H and13 C signals, two-dimensional 1 H, 1 H COSY and 1 H,13 C correlation spectra were recorded. Chemical shifts (δ) are given in parts per million relatives to the solvent residual peak. For clarity, the atom numbering used for NMR signals attribution is described in the figure1. The following abbreviations are used for multiplicity of NMR signals: s=singlet, d=doublet, t=triplet, q=quadruplet, m=multiplet, b=broad signal and app=apparent multiplicity. The given J values refer to apparent multiplicities and do not represent the true coupling constants. Mass and high-resolution mass spectrometry spectra were recorded on a Bruker microTOFQ apparatus. Compounds 2-4, 10, 18-20 and 22 have been prepared according to literature procedures.[11,47,52] 100 mg, 0.40 mmol) in a mixture of dry acetonitrile/dichloromethane 2/3 (v/v) (8 mL), 110 µL of DAST (2 eq., 0.81 mmol) were added dropwise at 0 ºC under inert atmosphere. The reaction mixture was stirred for 2 h at room temperature. The mixture was neutralized by addition of a saturated solution of NaHCO 3 (10 mL) and the organic solvent was removed under vacuum. The aqueous layer was extracted with CH 2 Cl 2 (2x20 mL) and the organic layer was dried over MgSO 4 .The solvent was removed under vacuum and the crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 60/40) to afford compound 6 (86 mg). Yield: 85 %; colorless oil; R f = 0.85 (cyclohexane/EtOAc 6/4); [α] D = -11.3 (c = 0.10, CHCl 3 ); IR (film): ν 2988, 2955, 1734, 1437, 1381,1307, 1267 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.34 (s, 3H, CH 3 ), 1.49 (s, 3H, CH 3 ), 2.72 (dd, 1H, J 2a,2b = 16.5 Hz, J 2a,3 = 7.0 Hz, H-2a), 2.78 (dd, 1H , J 2b,3 = 7.0 Hz, H-2b), 3.71 (s, 3H, OCH 3 ), 4.19 (app dt, 1H, J 6,F = 33.0 Hz, J 6,7a = J 6,7b = 3.0 Hz, H-6), 4.40-4.46 (m, 1H, H-3), 4.46 (ddd, 1H, J 7a,F = 47.5 Hz, J 7b,7a = 10.5 Hz, J 7b,6 = 3.5 Hz , H-7b), 4.58 (ddd, 1H, J 7b,F = 47.5 Hz, J 7b,7a = 10.5 Hz, J 7b,6 = 3.0 Hz, H-7b ), 4.80 (dd, 1H, J 4,5 = 6.0 Hz, J 4,3 = 4.0 Hz, H-4), 4.85 (app brd, 1H, H-5); 13 C NMR (CDCl 3 , 100.6 MHz): δ 25.1 (CH 3 ), 26.3 (CH 3 ), 36.4 (C-2), 51.9 (OCH 3 ), 78.6 (d, J C3-F = 2 Hz, C-3), 81.6 (C-4), 82.4 (d, J C5-F = 8 Hz, C-5), 82.5 (d, J C6-F = 19 Hz, C-6), 85.1 (d, J C7-F = 172 Hz, C-7), 112.9 (C(CH 3 ) 2 ), 171.6 (C=O). HRMS (ESI): calcd for C 11 H 18 FNaO 5 [M+Na] + : 271.0958, found: 271.0952.3,6-anhydro-2-deoxy-5-hydroxy-7-iodo-D-ribo-1,4-lactone 200 mg, 0,81 mmol) in dry THF (8 mL), 111 mg of imidazole (2.0 eq., 1.62 mmol) and 425 mg of triphenylphosphine (2.0 eq., 1.62 mmol) were added. The reaction was heated at reflux and 350 mg of I 2 (1.2 eq., 0.97 mmol) solubilized under inert atmosphere in 2 mL of dry THF were added. The reaction was stirred at reflux for 3 h. The solvent was evaporated and the crude intermediate 5 was used without further purification. The intermediate 5 was dissolved in a mixture of TFA/CH 2 Cl 2 4/1 (v/v) (10 mL) at 0 ºC. The reaction was stirred at room temperature for 3 h. The mixture was neutralized by addition of solid NaHCO 3 and diluted with water. The aqueous layer was extracted with CH 2 Cl 2 (3x20 mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum. The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 0/100) to afford compound 7 (154 mg). Yield: 67 %; white solid; R f = 0.30 (cyclohexane/EtOAc 7/3); [α] D = 49.9 (c = 0.11, CHCl 3 ); mp: 143-145ºC; IR (film): ν 3335, 2955, 2833, 1774, 1396, 1362, 1292 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.17 (d, 1H, J OH,5 = 10.5 Hz OH), 2.76 (dd, 1H, J 2a,2b = 19.0 Hz, J 2a,3 = 1.0 Hz, H-2a), 2.84 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.35 (dd, 1H, J 7a,7b = 12.0 Hz, J 7a,6 = 6.0 Hz, H-7a) , 3.51-3.57 (m, 2H, H-6, H-7b), 4.07 (ddd, 1H, J 5,6 = 8.0 Hz, J 5,4 = 4.5 HZ, H-5), 4.92 (ddd, 1H, J 3,4 = 4.5 Hz, H-3), 5.00 (app t, 1H, J 4,3 = J 4,5 = 4.5 Hz, H-4);13 C NMR (CDCl 3 , 100.6 MHz): δ 6.6 (C-7), 37.1 (C-2), 76.2 (C-3), 77.4 (C-5), 79.6 (C-6), 82.9 (C-4), 174.6 (C=O); HRMS (ESI): calcd for C 7 H 9 NaIO M+Na] + : 306.98443, found: 306.9438. 86 mg, 0.35 mmol) was dissolved in a mixture of TFA/CH 2 Cl 2 4/1 (v/v) (10 mL) at 0 ºC.The reaction was stirred at room temperature for 3 h. The mixture was neutralized by addition of solid NaHCO 3 and diluted with water. The aqueous layer was extracted with CH 2 Cl 2 (3x20 mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum. The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 0/100) to afford compound 8 (57 mg). Yield: 93 %; white solid; R f = 0.28 (cyclohexane/EtOAc 6/4); [α] D = 103.9 (c = 0.11, CHCl 3 ); mp: 93-95ºC; IR (film): ν 3377, 1737, 1357, 1292, 1200 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.26 (d, 1H, J OH,5 = 10.0 Hz, OH), 2.75 (brd, 1H, J 2a,2b = 19.0 Hz, H-2a), 2.85 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.87-3.95 (brdd, 1H, J 6,F = 27.0 Hz, J 6,5 = 9.0 Hz, H-6), 4.32 (ddd, 1H, J 5,6 = 9.0 Hz, J 5,4 = 4.5 Hz, H-5), 4.55 (ddd, 1H, J 7a,F = 47.5 Hz, J 7a,7b = 10.5 Hz, J 7a,6 = 3.5 Hz, H-7a), 4.68 (ddd, 1H, J 7b,F = 47.5 Hz, J 7b,6 = 1.5 Hz, H-7b), 4.91 (app brt, 1H, J 3,4 = 4.5 Hz, H-3), 4.99 (app t, 1H, J 4,3 = J 4,5 = 4.5 Hz, H-4). 13 C NMR (CDCl 3 , 100.6 MHz): δ 37.0 (C-2), 72.0 (d, J C5-F = 7 Hz, C-5), 76.5 (C-3), 80.8 (d, J C6-F = 18 Hz, C-6), 81.8 (d, J C7-F = 175 Hz, C-7) 82.7 (C-4), 174.8 (C=O); HRMS (ESI): calcd for C 7 H 9 NaFO 4 [M+Na] + : 199.0383, found: 199.0424.

J 5 , 6 = 8 . 5

 5685 Hz, J 5,4 = 4.5 Hz, H-5), 4.88 (ddd, 1H, J 3,2b = 6.0 Hz, J 3,4 = 4.5 Hz, J 3,2a = 1.0 Hz, H-3), 4.98 (app t, 1H, J 4,5 = J 4,3 = 4.5 Hz, H-4);13 C NMR (CDCl 3 , 100.6 MHz): δ 37.1 (C-2), 61.3 (C-7), 72.4 (C-5), 76.2 (C-3), 81.4 (C-6), 83.1 (C-4), 174.9 (C=O). HRMS (ESI): calcd for C 7 H 10 NaO 5

  crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford compound 13 (158 mg). Yield: 48 %; colorless oil; R f = 0.40 (cyclohexane/EtOAc 7/3); [α] D 25 22.4 (c = 0.06, CHCl 3 ); IR (film): ν 3672, 2926, 1782, 1472, 1427, 1394,1250 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Si-tert-butyl), 2.10 (brs, 1H, OH), 2.72 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 1.5 Hz, H-2a), 2.82 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.81 (dd, 1H, J 7a,7b = 11.0 Hz, J 7a,6 = 3.5 Hz, H-7a) , 3.85 (ddd, 1H, J 6,5 = 7.0 Hz, J 6,7a = 3.5 Hz, J 6,7b = 2.5 Hz, H-6), 3.91 (dd, 1H, J 7b,7a = 11.0 Hz, J 7b,6 = 2.5 Hz, H-7b), 4.40-4.46 (m, 1H, H-5), 4.89 (ddd, 1H, J 3,2b = 6.0 Hz, J 3,4 = 4.5 Hz, J 3,2a = 1.5 Hz, H-3), 5.01 (app t, 1H, J 4,3 = J 4,5 = 4.5 Hz, H-4), 7.37-7.46 (m, 6H, H Ar ), 7.65-7.69 (m, 4H, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz): δ 19.4 (C-Si), 27.0 (Si-tert-butyl), 37.2 (C-2), 63.5 (C-7), 73.0 (C-5), 76.5 (C-3), 82.9 (C-6), 83.3 (C-4), 127.9 (2CAr), 128.0 (2CAr), 130.0 (CAr), 130.0 (CAr), 133.1 (Cq Ar ), 133.1 (CqAr), 135.7 (2CAr), 135.7 (2CAr), 175.3 (C=O); HRMS (ESI): calcd for C 23 H 28 NaO 5 Si [M+Na] + : 435.1604, found: 435.1598.

  chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford 15 (106 mg). Yield: 88 %; colorless oil; R f = 0.25 (cyclohexane/EtOAc 7/3); [α] D 25 84.4 (c = 0.10, CHCl 3 ); IR (film): ν 3088, 2927, 2856, 1782, 1589, 1454, 1427, 1398, 1360, 1308, 1261 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.03 (s, 9H, Si-tert-butyl), 2.71 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 2.5 Hz, H-2a), 2.77 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.74 (dd, 1H, J 7a,7b = 11.5 Hz, J 7a,6 = 3.0 Hz, H-7a), 3.90 (dd, 1H, J 7b,6 = 2.5 Hz, H-7b), 4.06 (app dt, 1H, J 6,5 = 7.0 Hz, H-6), 4.26 (dd, 1H, J 5,4 = 4.5 Hz, H-5), 4.56 (d, 1H, J = 11.5 Hz, CH 2 Ph), 4.76 (d, 1H, CH 2 Ph), 4.86 (ddd, 1H, J 3,4 = 4.5 Hz, H-3), 4.98 (app t, 1H, H-4), 7.29-7.46 (m, 11H, H Ar ), 7.62-7.65 (m, 4H, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz): δ 19.4 (C-Si), 27.0 (Si-tert-butyl), 37.1 (C-2), 63.1 (C-7), 72.9 (CH 2 Ph), 77.1 (C-3), 78.2 (C-5), 81.4 (C-4), 81.9 (C-6), 127.9 (2CAr), 127.9 (2CAr), 128.1 (2CAr), 128.2 (2CAr), 128.7 (CAr), 129.9 (CAr), 130.0 (CAr), 133.1 (CqAr), 133.3 (CqAr), 135.7 (2CAr), 135.7 (2CAr), 137.4 (CqAr), 175.6 (C=O); HRMS (ESI): calcd for C 30 H 34 NaO 5 Si [M+Na] + : 525.2068, found: 525.2077.

7 (

 7 cyclohexane/EtOAc 80/20) to afford 17(10 mg). Yield: 61 %; white solid; R f = 0.25 (cyclohexane/EtOAc 7/3); [α] D 25 172.7 (c = 0.28, CHCl 3 ); mp: 80-82ºC; IR (film): ν 3343, 2920, 2870, 1770, 1452, 1394, 1310, 1259 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.69 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 1.0 Hz, H-2a), 2.78 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.65 (br d, 1H, J 7a,7b = 12.0 Hz, H-7a), 3.89 (dd, 1H, J 7b,6 = 2.0 Hz, H-7b), 3.93 (app dt, 1H, J 6,5 = 8.5 Hz, H-6), 4.08 (dd, 1H, J 5,4 = 4.5 Hz, H-5), 4.61 (d, 1H, J = 11.5 Hz, CH 2 Ph), 4.77 (d, 1H, CH 2 Ph), 4.84 (ddd, 1H, J 3,4 = 4.5 Hz, H-3), 4.93 (app t, 1H, H-4), 7.29-7.42 (m, 5H, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz): δ 37.0 (C-2), 61.2 (C-7), 72.8 (CH 2 Ph), 76.9 (C-3), 77.7 (C-5), 80.1 (C-6), 80.9 (C-4), 128.2 (2CAr), 128.4 (CAr), 128.2CAr), 137.3 (CqAr), 175.4 (C=O); HRMS (ESI): calcd for C 14 H 17 O 5 [M+H] + : 265.1071, found: 265.1057.

32 : 7 = 7 . 5

 32775 Yield: 15%; white solid; R f = 0.33 (toluene/acetone 8/2); [α] D = 23.1 (c = 0.16, CHCl 3 ); mp: 152-154ºC; IR (film): ν 3390, 2924, 2853, 1770, 1454, 1363, 1248, 1363, 1248 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.77 (d, 2H, J 2,3 = 5.0 Hz, H-2), 3.07-3.11 (m, 2H, OH-5 and OH-7), 3.94 (dd, 1H, J 6,Hz, J 6,5 = 5.0 Hz, H-6), 4.58 (app t, 1H, J 5,6 = J 5,4 = 5.0 Hz, H-5), 4.64 (app q, 1H, J 3,2 = J 3,4 = 5.0 Hz, H-3), 4.99 (app t, 1H, H-4), 5.02 (d, 1H, H-7), 7.33 (br d, 1H, J = 7.0 Hz, H Ar ), 7.38 (br t, 2H, J = 7.0 Hz, H Ar ), 7.43 (br d, 2H, J = 7.0 Hz, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz):  36.1 (C-2), 71.7 (C-5), 72.8 (C-7), 76.5 (C-3), 82.5 (C-4), 85.3 (C-6), 126.7 (2CAr), 128.4 (CAr), 128.7 (2CAr), 140.6 (CqAr), 174.9 (C=O); HRMS (ESI): calcd for C 13 H 14 NaO 5 [M+Na] + : 273.0733, found:

  Biological evaluationCytotoxic activity: MDA-MB231 Breast cancer cells, U87 and U251 glioblastoma cells and DAOY medulloblastoma cells were seeded in P96 plate at 5.10^4 cells per mL. 24 h post-incubation, cells were treated with different concentrations of lactone products (0.1; 1; 10; 100; 250 µM). Doxorubicin was used as reference. Then, MTT test was performed after 72 h of treatment adding 2.5 mg/ml MTT solution on cells. Formazan crystals have been dissolved in DMSO and results were obtained by an absorbance lecture at 540 nm. IC 50 was then calculated from fitting curves by linear regression with graphpad prism 8.Cells cycle:The detection of the different phases of the cell cycle is based on the fluorescence emission of propidium iodide. Propidium iodide is a fluorochrome (λex = 488 nm, λem = 617 nm) that intercalates between the bases of the DNA double helix. Thanks to this property, the fluorescence intensity is dependent on its DNA content. Two peaks of fluorescence can then be observed: cells in G1 phase with 2N chromosomes, cells in G2-M phase with 4N chromosomes and cells in S phase whose DNA content is between 2N and 4N. Experimentally, after three days of culture, U251 cells are treated with 1 µM lactones or 0.1 µM doxorubicin (positive control). After 72 h of contact with the compounds, the cells are recovered by trypsination and 1.10^6 cells of each condition are fixed in cold 70% ethanol. Then, half of this suspension (5.10^5 cells) is centrifuged for 10 minutes at 500 g and the pellet is resuspended in 500 µl of labelling buffer: PBS containing 0.5 mg/mL RNase A and 2.5 µg/ml propidium iodide. After 10 min of incubation in the dark in the labelling buffer, the cells are passed to the BD Accuri C6 Plus flow cytometer (BD biosciences) to measure the different phases of the cell cycle.Crystallographic data for the structure reported in this article have been deposited at the Cambridge Crystallographic Data Center and allocated with the deposition number CCDC 2083324 for compound 32.

  Cl 2 , Amberlite IR-120, AcOH/H 2 O 8/2). Nevertheless, the C-7 unprotected compound 12 was consistently obtained in quantitative yield. We moved on silyl protecting groups and O-TBDMS and O-TBDPS derivatives 10 and 11 were successfully prepared in good yields by reaction with the corresponding silyl chloride and imidazole in DMF. With PMB and TBDMS, the concommitant acetal deprotection and lactonization occurred but along with the unwanted removal of these protecting groups. The TBDPS group is known to be less acid sensitive than TBDMS and thus the compound 13 was successfully obtained in 36% yield when the reaction was performed in a mixture of AcOH/H 2 O (8/2) at 0°C, which were the best conditions to obtain 13. The simultaneous formation of compound 12 in 42% could not be avoided.

	The lactonization prior to the protection of hydroxyl in position 7 seemed to be a more efficient way
	in terms of global yield of compound 13. Compound 4 treated in a mixture of trifluoroacetic acid
	TFA/H 2 O (1/1) for 3 hours led to lactone 12 in 69 % yield and subsequent reaction with TBDPSCl
	and imidazole in DMF furnished 13 in 48 % yield, even when more equivalents of silylating agent
	were of used.
	7-O-
	PMB compound 9 was treated by a mixture of trifluoroacetic acid TFA/H 2 O (1/1) at 0 °C, which are
	the conditions used for lactonization of related compounds in our previous work. We only observed

the formation of the unprotected bicyclic compound 12 revealing the instability of the PMB protecting group in these acidic conditions. Other carefully controlled acidic conditions were attempted to perform lactonization while avoiding PMB removal (1N HCl in MeOH, TFA in CH 2

Table 1 .

 1 Scope and limitation of O-alkylation of 13

	Entry	Reagent	Base	Solvent	T
		(2 eq.)	(1 eq.)		(°C)

t (h) Starting compound 13 (%) Alkylated compound (%)

	of this diol by NaIO 4 led to aldehyde 21, used without purification in the susbsequent reduction to
	alcohol 22 or nucleophilic addition of phenyl magnesium bromide (Scheme 4).
	Starting from alcohol 22, the targeted 7-iodo derivative 24 was prepared by reaction with iodine
	followed by lactonization performed in a TFA/H 2 O (1/1 v/v) mixture. From compound 22, the
	lactonization in a TFA/H 2 O (1/1 v/v) mixture and the subsequent reaction with TBDPSCl likewise
	led to compound 26 in 57 % yield. According to conditions established for compound
	1	MeI	NaH	DMF	0	0.5	0	0
	2	MeI	NaH	DMF	-20	0.5	0	0
	3	Me 2 SO 4	K 2 CO 3	DMF	20	12	100	0
	4	BnBr	K 2 CO 3	DMF	20	12	100	0
	5	BnBr	K 2 CO 3	DMF	70	12	0	0
	6	BnBr	KHMDS	THF	-80	1	0	0
	7	BnBr	Ag 2 O a	CH 2 Cl 2	60 b	24	15	48
	8	BnBr	Ag 2 O a	CH 2 Cl 2	60 b	48	0	88
	9	allyBr	Ag 2 O a	CH 2 Cl 2	60 b	48	37	35
	10	MeI	Ag 2 O a	CH 2 Cl 2	60 b	48	0	72
	a : 3eq. of Ag 2 O, b: in sealed tube
	We next turned to the preparation of C-5/C-6 stereoisomers starting from commercially available D-
	-1,4-gulonolactone 18. As described above for compound 3, C-glycosyl compound 19 was obtained
	by a three steps synthetic sequence involving hydroxyl protection by isopropylidene, Wittig reaction
	and stereoselective hydrogenation. Selective 7,8-isopropylidene removal was carried out in a 1N HCl
	methanolic solution and diol 20 was thus obtained in 90 % yield (Scheme 3). The oxydative clivage

13, compound 26 was

  benzylated in the presence of Ag 2 O in CH 2 Cl 2 . Despite the low yield of this etherification (29 %), the expected 5-O-benzyl ether 28 was obtained after TBDPS removal which was carried out in presence of a catalytic amount of PTSA in MeOH.

Table 2 .

 2 IC 50 values of compounds 24, 35 and Doxorubicine

			IC 50 (µM) a
	Compounds	MDA-MB-231	DAOY	U87	U251
	35	534.6	612.4	32.40	1.341
	24	nd	141.07	7.606	528.6
	Doxorubicin	0.4817	0.0629	0.1864	0.106

a : The values are means of three independent experiments done in quadruplicates. Coefficients of variation are <10%.

  J 2a,2b = 18.5 Hz, J 2a,3 = 1.0 Hz, H-2a), 2.85 (dd, 1H, J 2b,2a = 18.5 Hz, J 2b,3 = 6.0 Hz, H-2b), 3.74 (dd, 1H, J 7a,7b = 12.0 Hz, J 7a,6 = 4.5 Hz, H-7a), 3.78 (ddd, 1H, J 6,5 = 8.5 Hz,

	(244 mg). Yield: 69 %; yellowish oil; R f = 0.40 (EtOAc/MeOH 9/1); [α] D 25 77.4 (c = 0.13, EtOH);
	IR (film): ν 3389, 2934, 2872, 1770, 1678, 1400, 1356, 1305, 1263, 1193 cm -1 ; 1 H NMR (CDCl 3 ,
	400 MHz): δ 2.73 (dd, 1H, J 6,7a = 4.5 Hz, J 6,7b = 2.5 Hz, H-6), 3.94 (dd, 1H, J 7b,7a = 12.0 Hz, J 7b,6 = 2.5 Hz, H-7b), 4.29 (dd, 1H,
		1.86 g). Yield: 95 %; colorless oil; R f =
	0.47 (cyclohexane/EtOAc 8/2); [α] D	25 0.40 (c = 0.36, CHCl 3 ); IR (film); ν 2928, 1742, 1425, 1351,
	1210 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Si-tert-butyl), 1.35 (s, 3H, CH 3 ), 1.49 (s, 3H,
	CH 3 ), 2.70 (dd, 1H, J 2a,2b = 16.5 Hz, J 2a,3 = 7.0 Hz, H-2a), 2.76 (dd, 1H, J 2b,3 = 7.0 Hz, H-2b), 3.67
	(dd, 1H, J 7a,7b = 11.0 Hz, J 7a,6 = 4.0 Hz, H-7a), 3.71 (s, 3H, COOCH 3 ), 3.77 (dd, 1H, J 7b,6 = 4.0 Hz,
	H-7b), 4.11 (app bt, 1H, H-6), 4.59 (app td, 1H, J 3,2a = J 3,2b = 7.0 Hz, J 3,4 = 4.0 Hz, H-3), 4.80 (dd,
	1H, J 4,5 = 6.0 Hz, H-4), 4.86 (br d, 1H, H-5), 7.36-7.46 (m, 6H, H Ar ), 7.64-7.68 (m, 4H, H Ar ); 13 C
	NMR (CDCl 3 , 100.6 MHz): δ 19.2 (C-Si), 25.2 (CH 3 ), 26.4 (CH 3 ), 27.0 (Si-tert-butyl), 34.9 (C-2),
	51.8 (OCH 3 ), 65.4 (C-7), 78.6 (C-3), 82.0 (C-4), 83.4 (C-5) , 84.3 (C-6), 112.5 (C(CH 3 ) 2 ), 128.0
	(4CAr), 130.0 (CAr), 130.0 (CAr), 132.9 (Cq Ar ), 133.0 (CqAr), 135.8 (4CAr), 171.8 (C=O); HRMS
	(ESI): calcd for C 27 H 36 KO 6 Si: [M+K] + : 523.1913, found: 523.1877.
	3,6-anhydro-2-deoxy-5,7-hydroxy-D-ribo-1,4-lactone 12

A solution of 4 (500 mg, 2.03 mmol in a mixture of TFA/H 2 O 1/1 (v/v) (20 mL) was stirred for 3 h at room temperature. The solvent was removed in vacuum and the crude residue was purified by column chromatography on silica gel (eluent: EtOAc/MeOH 100/0 to 90/10) to afford compound 12

  The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford 14 (74 mg).

	Yield: 72 %; colorless oil; R f = 0.72 (cyclohexane/EtOAc 6/4); [α] D 25 65.9 (c = 2.48, CHCl 3 ); IR
	(film) : ν 2930, 2857, 1782, 1462, 1390 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Si-tert-
	butyl), 2.69 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 2.0 Hz, H-2a), 2.77 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.47
	(s, 3H, OCH 3 ), 3.75 (dd, 1H, J 7a,7b = 11.5 Hz, J 7a,6 = 3.0 Hz, H-7a), 3.91 (dd, 1H, J 7b,6 = 2.5 Hz, H-
	7b), 3.98 (app dt, 1H, J 6,5 = 7.0 Hz, H-6), 4.07 (dd, 1H, J 5,4 = 4.5 Hz, H-5), 4.89 (ddd, 1H, J 3,4 = 4.5
	Hz, H-3), 5.07 (app t, 1H, H-4), 7.36-7.47 (m, 6H, H Ar ), 7.67 (br d, 4H, J = 6.5 Hz, H Ar ); 13 C NMR
	(CDCl 3 , 100.6 MHz): δ 19.4 (C-Si), 27.0 (Si-tert-butyl), 37.0 (C-2), 58.6 (CH 3 ), 63.3 (C-7), 77.1 (C-
	3), 80.7 (C-5), 81.2 (C-4), 81.8 (C-6), 127.9 (2CAr), 127.9 (2CAr), 130.0 (CAr), 130.0 (CAr), 133.2
	(CqAr), 133.3 (CqAr), 135.7 (2CAr), 135.7 (2CAr), 175.5 (C=O); HRMS (ESI): calcd for
	C 24 H 30 NaO 5 Si [M+Na]

+ : 449.1760, found: 449.1818.

-anhydro-2-deoxy-5,7-hydroxy-7-phenyl-D-gulo-1,4-lactone 31 and 32

  J 7a,7b = 12.0 Hz, J 7a,6 = 7.0 Hz, H-7a), 3.81 (dd, 1H, J 7b,6 = 4.5 Hz, H-7b), 3.92 (app dt, 1H, J 6,5 = J 6,7b = 5.0 Hz, H-6), 4.41 (app t, 1H, J 5,4 = J 5,6 = 5.0 Hz, H-5), 4.67 (ddd, 1H, J 3,4 = 5.0 Hz, H-3), 5.01 (app t, 1H, J 4,5 = J 4,3 = 5.0 Hz, H-4);13 C NMR (CD 3 OD, 100.6 MHz): δ 37.2 (C-2), 61.8 (C-7), 72.3 Butyldiphenylsilyl chloride (1.1 eq., 1.25 mmol) and 171 mg of imidazole (2.2 eq., 2.51 mmol) in dry DMF (2 mL) was added dropwise at 0 ºC under inert atmosphere. The reaction was stirred for 24 h at 40 ºC. The reaction mixture was diluted with water (15 mL) and extracted with EtOAc (3x20 mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under vacuum. 5.0 Hz, J 5,6 = 3.5 Hz, H-5), 4.47 (d, 1H, J = 11.5 Hz, CH 2 Ph), 4.69 (ddd, 1H, J 3,4 = 7.0 Hz, H-3), 4.78 (d, 1H, CH 2 Ph), 5.04 (dd, 1H, H-4), 7.23-7.45 (m, 11H, H Ar ), 7.62-7.66 (m, 4H, H Ar );13 C NMR H-5), 4.76 (dd, 0.35H, J 4,5 = 6.0 Hz, J 4,3 = 3.5 Hz, H-4), 4.79 (dd, 0.35H, H-5), 5.02 (t, 0.35H, J 7,OH = J 7,6 = 6.5 Hz, H-7), 5.07 (d, 0.65H, J 7,6 = 8.0 Hz, H-7), 7.27-7.53 (m, 5H, H Ar ). J 5,4 = J 5,OH = J 5,6 = 6.0 Hz, H-5), 4.72 (td, 1H, J 3,2a = J 3,4 = 6.0 Hz, J 3,2b = 3.5 Hz, H-3), 4.96 (app t, 1H, J 4,5 = J 4,3 = 6.0 Hz, H-4), 5.11 (dd, 1H, J 7,OH = 3.0 Hz, H-7), 7.30-7.40 (m, 3H,

	(CDCl 3 , 100.6 MHz): δ 19.3 (C-Si), 27.0 (Si-tert-butyl), 36.2 (C-2), 62.2 (C-7), 73.9 (CH 2 Ph), 76.0
	/80) to (C-5), 77.4 (C-3), 84.0 (C-6), 85.0 (C-4), 178.4 (C=O); HRMS (ESI): calcd for C 7 H 10 NaO 5 [M+Na] + : 197.0426, found: 197.0420. (C-3), 77.2 (C-5), 82.4 (C-4), 82.8 (C-6), 127.9 (4CAr), 128.0 (CAr), 128.2 (2CAr), 128.5 (2CAr), 129.9 (CAr), 129.9 (CAr), 133.4 (CqAr), 133.5 (CqAr), 135.7 (2CAr), 135.7 (2CAr), 137.5 (CqAr), 3,6Mixture 29/30 (180 mg, 0.56 mmol) was dissolved in a solution of TFA/H 2 O 6/4 (10 mL) and stirred
	provide 23 (326 mg). Yield: 61 %; colorless oil; R f = 0.60 (cyclohexane/EtOAc 5/5); [α] D = 15.6 (c = 175.3 (C=O); HRMS (ESI): calcd for C 30 H 35 O 5 Si [M+H] + : 503.2248, found: 503.2246. for 16 h at room temperature. The solvent was removed under vacuum and the crude product was
	0.10, CHCl 3 ); IR (film): ν 3333, 2988, 2939, 1774, 1400, 1362, 1281, 1248 cm -1 ; 1 H NMR (CDCl 3 , 3,6-anhydro-2-deoxy-5-hydroxy-7-O-(tert-butyldimethylsilyl)-D-galacto-1,4-lactone 26 purified by chromatography on silica gel (eluent: toluene/acetone 85/15) to afford compounds 31 (35
	400 MHz): δ 1.33 (s, 3H, CH 3 ), 1.45 (s, 3H, CH 3 ), 2.74 (dd, 1H, J 2a,2b = 17.0 Hz, J 2,3 = 6.5 Hz, H-To a solution of 25 (200 mg, 1.14 mmol) in dry DMF (5 mL), a solution of 321 µL of tert-3,6-anhydro-2-deoxy-5-O-(benzyl)-7-hydroxy-D-galacto-1,4-lactone 28 mg) and 32 (21 mg).
	2a), 2.80 (dd, 1H, J 2b,3 = 7.0 Hz, H-2b), 3.25 (dd, 1H, J 7a,7b = 9.5 Hz, J 7a,6 = 6.0 Hz, H-7a), 3.34 (dd, To a solution of compound 27 (35 mg, 0.07 mmol) in methanol (2 mL), 26 mg of PTSA 31: Yield: 25%; white solid; R f = 0.38 (toluene/acetone 8/2); [α] D = 2.0 (c = 0.32, CHCl 3 ); mp: 121-
	1H, J 7b,6 = 8.5 Hz, H-7b), 3.70 (s, 3H, OCH 3 ), 3.79 (app brt, 1H, H-6), 3.97 (app brt, 1H, H-3), 4.74-monohydrate (2.0 eq., 0.14 mmol) were added. After stirring at 30 ºC overnight, the solvent was 123ºC; IR (film): ν 3404, 292, 2852, 1772, 1456, 1369, 1163 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ
	4.79 (m, 2H, H-4, H-5); 13 C NMR (CDCl 3 , 100.6 MHz): δ -1.1 (C-7), 25.1 (CH 3 ), 26.0 (CH 3 ), 33.5 removed in vacuum and the crude product was purified by chromatography on silica gel (eluent: 2.55 (d, 1H, J OH,7 = 3.0 Hz, OH-7), 2.63 (d, 1H, J OH,5 = 6.0 Hz, OH-5), 2.81 (dd, 1H, J 2a,2b = 19.0 Hz,
	(C-2), 51.9 (OCH 3 ), 77.9 (C-3), 80.8, 81.3 (C-4, C-5), 82.1 (C-6), 112.8 (C(CH 3 ) 2 ), 171.4 (C=O); Cyclohexane/EtOAc 80/20) to afford 28 (9 mg). Yield: 49%; colorless oil; R f = 0.34 J 2a,3 = 6.0 Hz, H-2a), 2.87 (dd, 1H, J 2b,3 = 3.5 Hz, H-7b), 4.02 (app t, 1H, J 6,7 = J 6,5 = 5.0 Hz, H-6),
	HRMS (ESI): calcd for C 11 H 18 IO 5 [M+Na] + : 357.0193, found: 357.0215. The crude product was purified by flash chromatography on silica gel (eluent: cyclohexane/EtOAc (cyclohexane/EtOAc 2/8); [α] D = 102.1 (c = 0.11, CHCl 3 ); IR (film): ν = 3447, 2924, 2882, 1497, 4.29 (app q, 1H,
	100/0 to 70/30) to afford compound 26 (187 mg). Yield: 57%; colorless oil; R f = 0.32 1454, 1400, 1340, 1311 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.77 (d, 2H, J 2,3 = 4.5 Hz, H-2), 3.77
	3,6-anhydro-2-deoxy-5-hydroxy-7-iodo-D-gulo-1,4-lactone 24 (cyclohexane/EtOAc 7/3); [α] D = 10.8 (c = 0.11, CHCl 3 ); IR (film): ν 3447, 2930, 2857, 1774, 1472,
	A solution of compound 23 (200 mg, 0.56 mmol) in a mixture of TFA/H 2 O 1/1 (v/v) (1 mL) was 1427 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Si-tert-butyl), 2.71 (dd, 1H, J 2a,2b = 18.5 Hz,
	stirred for 3 h at room temperature. The solvent was removed in vacuum and the crude residue was J 2a,3 = 3.0 Hz, H-2a), 2.78 (dd, 1H, J 2b,3 = 6.5 Hz, H-2b), 3.09 (d, 1H, J OH,5 = 5.5 Hz, OH), 3.91 (dd,
	purified by column chromatography on silica gel (eluent: EtOAc/MeOH 100/0 to 90/10) to afford 24 1H, J 7a,7b = 10.5 Hz, J 7a,6 = 4.5 Hz, H-7a), 3.95 (app q, 1H, J 6,7a = J 6,7b = J 6,5 = 4.5 Hz, H-6), 4.03 (dd, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz): δ 36.7 (C-2), 62.0 (C-7), 73.4 (CH 2 Ph), 76.6 (C-3), 78.5 (C-5),
	(70 mg). Yield: 44 %; white solid; R f = 0.33 (EtOAc/MeOH 9/1); [α] D = 49.6 (c = 0.10, CHCl 3 ); mp: 1H, H-7b), 4.51 (app dt, 1H, J 5,OH = J 5,4 = 5.5 Hz, H-5), 4.89 (ddd, 1H, J 3,4 = 5.5 Hz, H-3), 4.96 (app 81.4 (C-6), 81.7 (C-4), 128.2 (2CAr), 128.5 (CAr), 128.8 (2CAr), 136.9 (CqAr), 175.3 (C=O);
	183-185ºC; IR (film): ν 3333, 2988, 2939, 1774, 1400, 1362, 1281, 1248 cm -1 ; 1 H NMR (CDCl 3 , 400 t, 1H, H-4), 7.36-7.47 (m, 6H, H Ar ), 7.66-7.70 (m, 4H, H Ar ); 13 C NMR (CDCl 3 , 100.6 MHz): δ 19.2 HRMS (ESI): calcd for C 14 H 16 NaO 5 [M+Na] + : 287.0890, found: 287.089.
	MHz): δ 2.76 (dd, 1H, J 2a,2b = 19.0 Hz, J 2a,3 = 3.5 Hz, H-2a), 2.81 (dd, 1H, J 2b,3 = 6.0 Hz, H-2b), 3.26 (C-Si), 26.9 (Si-tert-butyl), 36.3 (C-2), 62.6 (C-7), 72.2 (C-5), 76.1 (C-3), 82.2 (C-6), 82.9 (C-4),
	(dd, 1H, J 7a,7b = 10.0 Hz, J 7a,6 = 6.5 Hz, H-7a), 3.42 (dd, 1H, J 7b,6 = 7.0 Hz, H-7b), 4.09 (app td, 1H, 127.9 (2CAr), 127.9 (2CAr), 130.0 (2CAr), 132.8 (Cq Ar ), 133.0 (CqAr), 135.7 (2CAr), 135.8 (2CAr), Compounds 29/30
	J 6,5 = 4.0 Hz, H-6), 4.49 (br dd, 1H, J 5,4 = 5.5 Hz, H-5), 4.73 (app td, 1H, J 3,2b = J 3,4 = 6.0 Hz, H-3), 175.1 (C=O); HRMS (ESI): calcd for C 23 H 28 KO 5 Si [M+Na] + : 451.1338, found: 451.1339. To a solution of intermediate aldehyde 21 (532 mg, 2.18 mmol) in dry Et 2 O (100 mL), were added
	5.04 (app t, 1H, H-4); 13 C NMR (CDCl 3 , 100.6 MHz): δ -0.8 (C-7), 35.9 (C-2), 71.0 (C-5), 76.6 (C-dropwise at -20 ºC, 2.15 mL of 1M phenyl magnesium bromide in THF (0.95 eq., 2.07 mmol). The
	3), 82.6 (C-4), 83.6 (C-6), 174.8 (C=O). HRMS (ESI): calcd for C 7 H 9 NaIO 5 [M+Na] + : 306.9438, 3,6-anhydro-2-deoxy-5-O-(benzyl)-7-O-(tert-butyldiphenylsilyl)-D-galacto-1,4-lactone 27 mixture was stirred overnight at room temperature. The reaction was quenched by addition of a
	found: 306.9448. To a solution of 26 (100 mg, 0.24 mmol) in dry dichloromethane (5 mL), 185 mg of Ag 2 O (3 eq., saturated solution of NH 4 Cl (until pH = 6) and the aqueous layer was extracted with Et 2 O (2x50 mL)
	0.73 mmol) and benzyl bromide (4 eq., 0.96 mmol) were added under inert atmosphere. The reaction and with CH 2 Cl 2 (2x50 mL). The combined organic layer was dried over MgSO 4 , filtered and
	3,6-anhydro-2-deoxy-5,7-hydroxy-D-gulo-1,4-lactone 25 was stirred for 48 h at 60 ºC in a sealed tube. The reaction mixture was filtered off through a Celite ® evaporated under vacuum. The crude residue was purified by column chromatography to provide a
	A solution of 22 (402 mg, 1.63 mmol) in a mixture of TFA/H 2 O 1/1 (v/v) (20 mL) was stirred for 3 h pad and the solvent was removed under vacuum. The crude product was purified by flash mixture of 29 and 30 (246 mg). Yield: 35%; colorless oil; R f = 0.32 (cyclohexane/EtOAc 6/4); 1 H
	at room temperature. The solvent was removed in vacuum and the crude residue was purified by chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford 27 (35 mg). NMR of 29/30 (ratio 65/35) (CDCl 3 , 400 MHz): δ 1.27 (s, 1.95H, CH 3 ), 1.35 (s, 1.05H, CH 3, ), 1.43
	column chromatography on silica gel (eluent: EtOAc/MeOH 100/0 to 90/10) to afford 25 (176 mg). Yield: 29%; colorless oil ; R f = 0.66 (cyclohexane/EtOAc 7/3); [α] D = 30.7 (c = 0.11, CHCl 3 ); IR (s, 1.95H, CH 3, ), 1.57 (s, 1.05H, CH 3, ), 2.79-2.91 (m, 2H, H-2), 3.57 (dd, 0.65H, J 6,7 = 8.0 Hz, J 6,5 =
	Yield: 62%; white solid; R f = 0.40 (EtOAc/MeOH 9/1); [α] D 25 = 58.7 (c = 0.10, EtOH); mp: 97-99ºC;
	IR (film): ν 3395, 2938, 1755, 1678, 1429, 1367, 1312, 1276 cm -1 . 1 H NMR (CD 3 OD, 400 MHz): δ
	2.61 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 2.5 Hz, H-2a), 2.83 (dd, 1H, J 2b,3 = 7.5 Hz, H-2b), 3.69 (dd, 1H,

(film): ν 2928, 2856, 1780, 1471, 1427 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 1.06 (s, 9H, Si-tertbutyl), 2.64 (dd, 1H, J 2a,2b = 18.5 Hz, J 2a,3 = 4.0 Hz, H-2a), 2.72 (dd, 1H, J 2b,3 = 7.5 Hz, H-2b), 3.83 (br dd, 1H, J 7a,7b = 12.5 Hz, J 7a,6 = 8.0 Hz, H-7a), 3.97-4.03 (m, 2H, H-6 and H-7b), 4.15 (dd, 1H, J 5,4 = (dd, 1H, J 7a,7b = 12.0 Hz, J 7a,6 = 5.0 Hz, H-7a), 3.81 (dd, 1H, J 7b,6 = 5.0 Hz, H-7b), 4.09 (dt, 1H, J 6,5 = 5.5 Hz, H-6), 4.29 (app t, 1H, J 5,4 = J 5,6 = 5.5 Hz, H-5), 4.50 (d, 1H, J = 11.5 Hz, CH 2 Ph), 4.72 (dt, 1H, J 3,4 = 5.5 Hz, H-3), 4.84 (d, 1H, J = 11.5 Hz, CH 2 Ph), 5.00 (app t, 1H, H-4), 7.30-7.39 (m, 5H, 3.5 Hz, H-6), 3.62 (dd, 0.35H, J 6,7 = 6.5 Hz, J 6,5 = 3.5 Hz, H-6), 3.68 (s, 1.05H, OCH 3 ), 3.72 (s, 1.95H, OCH 3 ), 3.90-3.99 (m, 1H, H-3), 4.38 (dd, 0.65H, J 4,5 = 6.0 Hz, J 4,3 = 3.5 Hz, H-4), 4.71 (dd, 0.65H, H Ar ), 7.47 (d, 2H, J = 7.5 Hz, H Ar ). 13 C NMR (CDCl 3 , 100.6 MHz): δ 36.2 (C-2), 71.8 (C-5), 72.3 (C-7), 76.2 (C-3), 82.5 (C-4), 86.1 (C-6), 126.9 (2CAr), 128.4 (CAr), 128.7 (2CAr), 140.2 (CqAr), 175.2 (C=O); HRMS (ESI): calcd for C 13 H 14 NaO 5 [M+Na] + : 273.0733, found: 273.0733.

  The crude was purified by chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 70/30) to afford 265 mg of esterified compounds 33/34 (53 % yield). A solution of 33/34 (80 mg, 0.18 mmol) in a mixture of TFA/CH 2 Cl 2 1/1 (v/v) (5 mL) was stirred under inert atmosphere for 24 h at room temperature. The solvent was removed under vacuum and the crude was purified by chromatography on silica gel (eluent: Et 2 O/petroleum ether 50/50 to 80/20) to afford compound 35 (125 mg). Yield: 56%; yellowish solid; R f = 0.22 (Et 2 O/petroleum ether 8/2); [α] D = 17.5 (c = 0.10, CHCl 3 ); mp: 157-159ºC; IR (film): ν 3416, 2920, 1770, 1697, 1634, 1450, 1371, 1269 cm -1 ; 1 H NMR (CDCl 3 , 400 MHz): δ 2.82 (dd, 1H, J 2a,2b = 19.0 Hz, J 2a,3 = 7.0 Hz, H-2a), 2.90 (dd, 1H, J 2b,3 = 3.0 Hz, H-2b), 4.14 (app t, 1H, J 5,4 = J 5,6 = 6.0 Hz, H-5), 4.27 (dd, 1H, J 6,7 = 7.5 Hz, H-6), 4.78 (app td, 1H, J 3,2a = J 3,4 = 6.0 Hz, H-3), 4.97 (app t, 1H, J 4,5 = J 4,3 = 6.0 Hz, H-4), 6.22 (d, 1H, H-7), 6.55 (d, 1H, J = 16.0 Hz, H-styryl), 7.29-7.43 (m, 6H, H Ar ), 7.48-7.56 (m, 4H, H Ar ), 7.70 (d, 1H, H-styryl). 13 C NMR (CDCl 3 , 100.6 MHz): δ 36.3 (C-2), 71.3 (C-5), 74.3 (C-7), 76.5 (C-3), 82.3 (C-4), 84.8 (C-6), 117.9 (C-styryl), 127.6 (2CAr), 128.4 (2CAr), 128.7 (2CAr), 128.8 (CAr), 129.0 (2CAr), 130.5 (CAr), 134.5 (CqAr), 137.4 (CqAr), 145.9 (C-styryl), 166.1 (C=O), 175.2 (C=O). HRMS (ESI): calcd for C 22 H 20 KO 6 [M+K] + : 419.0891, found: 419.0877.

Acknowledgments

This work was supported by Agence Nationale de la Recherche (grant: ANR-17-CE18-0018-03). The authorsgratefully acknowledge the University of Lorraine, CNRS, andthe European Regional Development Funds (Programme opérationnel FEDER-FSE Lorraine et Massif des Vosges 2014-2020/"Fire Light"project:"Photo-bio-active moleculesand nanoparticles") forfinancial support. We thank François Dupire and the MassLor Platform (Université de Lorraine, Nancy, France) for mass spectrometry. We thank NMR and PMD2X Platforms of the Jean Barriol Institute (Université de Lorraine, Nancy, France) for NMR and X-ray diffraction measurements, respectively.


Canestrini's Goby

Canestrinii Gobius

Ninni

( Teleostei

Gobiiformes

Gobiidae

Harald Gobionellinae

Ahnelt

Christelle Tougard

Marcelo Kovačić

Keywords: Gobiidae, Gobius canestrinii, taxonomy, lectotype, Venice, Italy zoobank.org 2E8EA3A1-6ED5-4CE6-9FE5-E7AB14201451

A recent study based on molecular biological data revealed that Gobius canestrinii (NINNI, 1883), a gobioid fish endemic to the Adriatic basin, has to be split in three lineages.

The original description was based on populations from two distant localities in Italy and in Croatia respectively, the first representing one of these lineages, the second most likely belonging to another lineage. To fix the name of the species, we hereby designated a lectotype for Canestrini's Goby.

INTRODUCTION

A recent study based on molecular data and species delimitation test revealed three lineages of Gobius canestrinii [START_REF] Ninni | Nuova specie di Gobius[END_REF], two of them cryptic species [START_REF] Tougard | Quaternary climatic cycles promoted (re)colonization and diversification events in Adriatic sand gobies[END_REF]. Nevertheless, as only three populations along the range of G. canestrinii were investigated, it is possible that further distinct lineages will be detected. Additionally, Canestrini's Goby seemingly represents a non-monophyletic assemblage of freshwater gobies with at least one of the lineages closely related to Pomatoschistus montenegrensis [START_REF] Miller | Pomatoschistus canestrinii (Ninni, 1883)[END_REF]Šanda, 2008 (TOUGARD et al., 2021).

The description of Canestrini's Goby was based on samples from two distant localities, one in Italy, Venice lagoon and one in Croatia, river Jadro near Split [START_REF] Ninni | Nuova specie di Gobius[END_REF]. The material from Italy was collected by A.P. Ninni, while the Croatian material was sent to him by J. Kolombatović. NINNI (1883) did not mention an exact number of specimens on which the original description of G. canestrinii was based. There is also no indication that one of the specimens would have to be addressed as a holotype. Therefore, all specimens have to be considered as syntypes.

To date twelve syntypes are documented: five at the Natural History Museum in Vienna (NMW) and seven at the Zoological Museum "La Specola" of the University of Florence (MZUF) [START_REF] Fricke | Eschmeyer's Catalog of Fishes: Genera, Species, References[END_REF]. The five syntypes housed at the NMW (register numbers NMW 28817-28818 and NMW 29943-29945) arrived there before May 1882. Ninni sent these specimens to F. Steindachner, head of the Ichthyological Collection of the K. K.

Naturhistorisches Hofmuseum in Vienna, who mentioned in a letter from May 6, 1882 that the new species would be a variety of Gobius quagga Heckel, 1839[START_REF] Ninni | Nuova specie di Gobius[END_REF]. The seven syntypes deposited at MZUF (register numbers MZUF 5577-5583) are dated "22. IV.1883[START_REF] Vanni | Cataloghi del Museo Zoologico "La Specola" dell[END_REF]. All 12 specimens were collected at Venice lagoon [START_REF] Ninni | Nuova specie di Gobius[END_REF]. The whereabouts of the type series from the Jadro River (Croatia) is unknown.

The study of [START_REF] Tougard | Quaternary climatic cycles promoted (re)colonization and diversification events in Adriatic sand gobies[END_REF] revealed that the population of the Canestrini's Goby from Italy and the two populations from Croatia represent three distinct species. To avoid taxonomical problems, a lectotype for G. canestrinii was designated.

Description of the lectotype

According to the International Code of Zoological Nomenclature (Art. 74.1,74.7 and amendment of Art. 74.7.3), a lectotype is herein designated to become the unique bearer of the name G. canestrinii. To fix the name of the species, we hereby designate a lectotype for Canestrini's Goby. The specimen is properly labeled at the Ichthyological Collection of the NMW and can be identified by its morphological characters described below. As the Venetian specimens of MZUF are all juvenile, an adult male registered under number NMW 29943, was selected as lectotype (Fig. 1). It is characterized by: -standard length + caudal fin length (SL + CL) of the lectotype 37.4 +7.2 mm; head length 9.7% SL; snout to origin of first dorsal fin 34.5% SL; snout to origin of second dorsal fin 55.1% SL; snout to origin of anal fin 57.5% SL; body depth at origin of pelvic fin 18.4% SL; body depth at origin of anal fin 16.3% SL; caudal peduncle length 26.7 % SL; caudal peduncle depth 1.0% SL; eye diameter (horizontal) 6.8% SL; snout length 7.0% SL; postorbital length 12.7% SL; -first dorsal fin with six spiny rays (D1 VI); second dorsal fin with one spiny and nine soft rays (D2 I/9); anal fin with one spiny and eight soft rays (A I/8); pectoral fin with 16 soft rays (P 16); ventral disc, formed by the fused pelvic fins, with one spiny and five soft rays on both sides, the fifth rays of both sides fused with each other along the entire length (V I/5+5/I). The ventral disc is complete with a distinct membrane expending between both spiny rays; the edge of this membrane is smooth; -all scales are ctenoid, increasing in size posteriorly; scales are largest on the caudal peduncle; ctenii are small and numerous (21-30); 34 scales in lateral midline; trunk anterior to a line of the axilla rearwards to the fourth fin ray of the second dorsal fin (D2 4) naked; head, nape, pre-dorsal area and breast naked; -head lateral line canal system with anterior oculo-scapular canal (AOS) present; following AOS, pores are developed: B, C (unpaired), D (unpaired), F and H; pore E is missing on both sides; posterior oculo-scapular canals (POS) reduced but furrows in the canal bearing bone (post-temporal) still distinct; pre-opercular canal (POC) present on the left side with pores M and O present; pore N missing; POC on the right side missing but furrow in the canal bearing bone (pre-opercle) still distinct;

-free neuromasts and their pattern not discernable because of preservation; -urogenital papilla elongate and pointed, wide at the base and tapering to the end; -coloration (preserved): body pale fawn; dorsal side of head and trunk brownish-greyish; indistinct pale blotch dorsal of each opercle but not confluent in the midline; sides of head (cheeks and opercles) and the trunk covered by numerous blackish spots; spots on nape and pre-dorsal area distinctly smaller except for two spots immediately anterior to the origin of the first dorsal fin; four such blackish spots extend as an oblique row on the anterior most part of the pectoral fin; pale mark on base on the origin of the pectoral fin rays anterior to these spots; first dorsal fin with a large, distinct blackish spot which is surrounded by transparent fin membrane; blotch extends from fifth spiny ray on to the post-dorsal membrane.

With the designation of the specimen NMW 29943 as lectotype of G. canestrinii the four syntypes registered NMW 28817-28818 and NMW 29944-29945 become paralectotypes (recommendation of ICZN Art. 47.F).

DISCUSSION

Formerly placed in the genus Pomatoschistus Gill, 1863[START_REF] De | Sur une collection de Gobiinae provenant du Maroc. Essai de synopsis des espèces de l'Europe[END_REF], G. canestrinii was recently transferred to the genus Ninnigobius Whitley, 1951[START_REF] Thacker | Phylogeny, systematics and biogeography of the European sand gobies (Gobiiformes: Gobionellidae)[END_REF]), whereas TOUGARD et al. (2021) found no clear evidence to assign G. canestrinii to this genus. However, G. canestrinii is endemic to the Adriatic drainage area and has a disjunct distribution [START_REF] Miller | Pomatoschistus canestrinii (Ninni, 1883)[END_REF]. Divers morphological variations were described on the Croatian samples of the supposed single species of G. canestrinii [START_REF] Kovačić | Morphological variability of Pomatoschistus canestrinii (Gobiidae), with the reduction of squamation and head canals[END_REF]. Based on molecular biological data, [START_REF] Tougard | Quaternary climatic cycles promoted (re)colonization and diversification events in Adriatic sand gobies[END_REF] split G. canestrinii in three lineages of which one corresponds to one of the type localities of G. canestrinii, the lagoon of Venice in northern Italy. The other two are lineages from Croatia, each one from a single locality. The river Raša lineage in Istria (west Croatia) is more close to the type locality in the Venice lagoon than to the second type locality, river Jadro near Split. The Baćinska lakes lineage is south from this second type locality, situated in southeast Croatia [START_REF] Miller | Pomatoschistus canestrinii (Ninni, 1883)[END_REF][START_REF] Kovačić | Morphological variability of Pomatoschistus canestrinii (Gobiidae), with the reduction of squamation and head canals[END_REF]. In order to fix the name of the species, according to the recommendation of the International Code of Zoological Nomenclature (Art. 74.1, 74.7) (International Commission on Zoological [START_REF] Nomenclature | [END_REF][START_REF]Declaration 44 -Amendment of Article 74.7.3[END_REF], one specimen, among the Venetian syntypes of G. canestrinii, was chosen as lectotype. The Jadro population, which should be distinct from the Venice population considering the Raša lineage and the Baćinska lakes lineage in between, is left as an undescribed species, probably related to the Baćinska lakes lineage.

Fig. 1 .

 1 Fig. 1. Lectotype of Gobius canestrinii (NMW 29943). Lateral view. Scale bar: 10 mm.

ACKNOWLEDGEMENTS

We thank Bettina Riedel (NMW) for her help with the type series of Gobius canestrinii and Nina Bogutskaya (NMW) for nomenclatorial advice.