pharmaceuticals

Article Total SynthesisSynthesis ofof NaturalNatural DisaccharideDisaccharide SambubioseSambubiose

, Simone Lucarini *,††, Maria, Maria Gessica Gessica Ciulla Ciulla †, Paola† , Paola Mestichelli Mestichelli and Andrea and Andrea Duranti Duranti * *

Department of Biomolecular Sciences, University of Urbino Carlo Bo Piazza del Rinascimento, 6,6, 61029 Urbino,61029 Urbino, Italy; [email protected] Italy; [email protected] (M.G.C.); (M.G.C.); [email protected] [email protected] (P.M.) (P.M.) * Correspondence: Correspondence: [email protected] (S.L.); [email protected] (A.D.);(A.D.) Tel.:Tel.: +39-0722-303333+39-0722-303333 (S.L.); (S.L.); +39-0722-303501+39-0722-303501 (A.D.) (A.D.) These authors contributed equally to this work. † These authors contributed equally to this work.


Received: 23 23 June 2020; Accepted: 13 13 Augu Augustst 2020; 2020; Published: Published: 17 17 August August 2020 2020


Abstract: AA practical practical and and robust robust synthetic synthetic method method to toob obtaintain the the natural natural disaccharide disaccharide sambubiose sambubiose (2- (2-O-βO--Dβ-xylopyranosyl--D-xylopyranosyl-D-glucopyranose)D-glucopyranose) is reported, is reported, exploring exploring the the key key step step in in the the synthesis, synthesis, i.e., i.e., stereoselective OO-glycosylation.-glycosylation. Specifically, Specifically, the the best combinationsbest combinations of glycoside of glycoside donors anddonors acceptors and wereacceptors identified, were identified, stereospecific stereosp controlecific of the control reaction of the was reaction achieved was by achieved screening by several screening catalysts several and catalystsprotection and/deprotection protection/deprotection steps were evaluated steps andwere improved. evaluated The and best improved. result was obtainedThe best byresult coupling was obtainedallyl 3,5,6-tri- by Ocoupling-benzyl- β-D-glucofuranosideallyl 3,5,6-tri-O-benzyl- withβ-D 2,3,4-tri--glucofuranosideO-acetyl-D-xylopiranosyl- with 2,3,4-tri-Oα-acetyl--trichloroD- xylopiranosyl-acetimidate inα the-trichloro presence acetimidate of trimethylsilyl in the triflate presence as aof catalyst trimethylsilyl giving the triflate corresponding as a catalyst protected giving thetarget corresponding compound as protected a correct target single compound isomer. The as latter a correct was single transformed isomer. accordingly The latter was into transformed the desired accordinglyfinal product into by deprotectionthe desired final steps product (deallylation, by dep deacetylation,rotection steps and(deallylation, debenzylation). deacetylation, Sambubiose and wasdebenzylation). synthesized Sambubiose into a satisfactory was synthesized and higher into overall a satisfactory yield than and previously higher overall reported yield and than was previouslyalso characterized. reported and was also characterized.

Keywords: chemopreventive phytochemicals; flavonoids;flavonoids; 2-O-xylosylvitexin; antiproliferative activity; carbohydrates; glycosylation;glycosylation; sambubiose; total synthesis

1. Introduction Introduction Chemopreventive phytochemicals phytochemicals (CPs) (CPs) occur occur naturally naturally in in some some plants plants and and have have been been shown shown to inhibitto inhibit all alllevels levels of carcinogenesis of carcinogenesis in both in both in viintro vitro andand in invivo vivo modelsmodels [1]. [1 Among]. Among CPs, CPs, flavonoid flavonoid 2- O2--xylosylvitexinO-xylosylvitexin (Figure (Figure 11),), which which is constitutedis constituted by carbohydrate by carbohydrate (sambubiose, (sambubiose, Figure1 )Figure and aglycone 1) and (apigenin,aglycone (apigenin, Figure1) Figure components, 1) componen was identifiedts, was identified for the firstfor the time first in timeCitrus in Citrus sinensis sinensisleaves leaves [2] and [2] andthen then in Beta in Beta vulgaris vulgarissub. sub.cycla cyclaleaves leaves and and seeds seeds [3] with [3] with the latterthe la containingtter containing the mostthe most abundant abundant and andefficacious efficacious CPs. CPs.

OH O

HO O OH OH O O O OH HO HO O O OH HO HO O HO O OH HO O OH OH OH OH Sambubiose Apigenin

OH OH 2-O-Xylosylvitexin Figure 1. ChemicalChemical structures structures of sambubiose, 2- O-xylosylvitexin, and apigenin.

Pharmaceuticals 2020, 13, 198;x; doi: doi: FOR10.3390 PEER/ph13080198 REVIEW www.mdpi.com/journal/pharmaceuticalswww.mdpi.com/journal/pharmaceuticals PharmaceuticalsPharmaceuticals 20202020,, 1313,, 198x FOR PEER REVIEW 2 2 ofof 1312

2-O-xylosylvitexin shows strong antiproliferative activity against human colon cell cancer lines 2-O-xylosylvitexin shows strong antiproliferative activity against human colon cell cancer lines while also enhancing apoptosis, increasing cells in the G1 phase and reducing cells in the S phase as while also enhancing apoptosis, increasing cells in the G1 phase and reducing cells in the S phase as well as the proliferation of human fibroblasts [4]. well as the proliferation of human fibroblasts [4]. Sambubiose, on the other hand, is formed by glucopyranose and xylopyranose units linked Sambubiose, on the other hand, is formed by glucopyranose and xylopyranose units linked through a 1,2-trans bond in 2-position and was first isolated as a component of anthocyanins from through a 1,2-trans bond in 2-position and was first isolated as a component of anthocyanins from elderberry (Sambucus nigra) [5], a plant with therapeutic properties [6,7]. In addition, sambubiose is elderberry (Sambucus nigra)[5], a plant with therapeutic properties [6,7]. In addition, sambubiose is also a constituent of the other major anthocyanins from black raspberries (Rubus occidentalis L.) [8], also a constituent of the other major anthocyanins from black raspberries (Rubus occidentalis L.) [8], red red currants, Davidson’s plums, and small red beans, and is linked to minor anthocyanins from açai currants, Davidson’s plums, and small red beans, and is linked to minor anthocyanins from açai and and Vaccinium padifolium blueberries [9]. Vaccinium padifolium blueberries [9]. To date, to the best of our knowledge, only one synthesis of sambubiose has been reported [10]; To date, to the best of our knowledge, only one synthesis of sambubiose has been reported [10]; however, the method followed by Erbing and Lindberg [10] (Scheme 1) and subsequently applied by however, the method followed by Erbing and Lindberg [10] (Scheme1) and subsequently applied by Dick et al. [11] has several drawbacks and improvements could be made in the reagents and Dick et al. [11] has several drawbacks and improvements could be made in the reagents and conditions. conditions.

SchemeScheme 1.1. Reagents and conditions: ( i) NaOH, BnCl, reflux, reflux, 7 h, 78%; (ii, iii) BnOH,BnOH, HCl,HCl, reflux,reflux, 44 hh thenthen AcAc22O, pyridine, rt, 24 h, 6%; (iv) MeONa, MeOH,MeOH, rt,rt, 1212 h,h, 22%;22%; ((vv,, vivi)) Hg(CN)Hg(CN)22//HgBrHgBr22, MeCN, MeCN, rt, 44 hh thenthen MeONa,MeONa, MeOH,MeOH, rt,rt, 1212 h,h, 54%;54%; ((viivii)H) H22, PdPd/C/C 10%, EtOH, rt, 2222 h,h, 43%.43%.

Firstly,Firstly, the the purification purification of of benzyl benzyl 3,5,6-tri- 3,5,6-tri-O-benzyl-O-benzyl-α-Dα-glucofuranoside-D-glucofuranoside (4a) (to4a eliminate) to eliminate high- high-boilingboiling benzyl benzyl alcohol alcohol from the from reaction the crude reaction mixture crude was mixture extremely was difficult extremely both dibyffi distillationcult both byand/or distillation by chromatography. and/or by chromatography.Erbing and Lindberg Erbing [10], therefore, and Lindberg had to [10employ], therefore, acetylation had and to employdeacetylation acetylation as additional and deacetylation synthetic assteps additional via benzyl synthetic 2-O steps-acetyl-3,5,6-tri- via benzylO-benzyl- 2-O-acetyl-α-D- 3,5,6-tri-glucofuranosideO-benzyl- (3aα)-D-glucofuranoside to purify 4a (Scheme (3a 1,) “iii” to purify and “iv”4a (Schemesteps). Secondly,1, “iii” and the “iv” use of steps). the intrinsically Secondly, theunstable use of donor the intrinsically 2,3,4-tri-O-acetyl- unstableα- donorD-xylopyranosyl 2,3,4-tri-O -acetyl-bromideα-D-xylopyranosyl (5a), which must bromidebe freshly (5a prepared), which mustand recrystallized be freshly prepared because and of recrystallized its low stability, because even of its when low stability,refrigerated, even whenwas also refrigerated, problematic. was alsoMoreover, problematic. Erbing Moreover,and Lindberg Erbing [10] and used Lindberg a stoichiometric [10] used a amount stoichiometric of mercuric amount cyanide of mercuric as a cyanideglycosylation as a glycosylation promoter. promoter.This reactant This reactantshould shouldnot be notused be usedbecause because of its of itshigh high toxicity toxicity and dangerousness.dangerousness. Furthermore, Hg(CN)Hg(CN)22 does not allow a stereospecificstereospecific controlcontrol ofof glycosylation:glycosylation: 6 [[12]12] mustmust bebe separatedseparated from from its its anomer anomer since since the the output output of theof the reaction reaction is an is αan- and α- andβ-mixture. β-mixture. Finally, Finally, and importantly,and importantly, the authors the authors did not did fully not characterize fully characterize the natural the disaccharide natural disaccharide sambubiose sambubiose [10]. Hence, [10]. the aimHence, of this the investigation aim of this investigation was to develop was an to improved develop eanffi cientimproved strategy efficient to synthesize strategy our to targetsynthesize molecule our overcomingtarget molecule theabovementioned overcoming thedrawbacks. abovementioned The key drawbacks. synthetic step The (i.e., key the syntheticO-glycosylation step (i.e., between the O- theglycosylation appropriate between protected the glucose appropriate and activated protected/protected glucose and xylose) activated/protected must be thoroughly xylose) explored. must Inbe particular,thoroughly the explored. leaving groupIn particular, of the donor the leaving should group be screened, of the donor and the should correct be catalystscreened,/promoter and the shouldcorrect becatalyst/promoter fine-tuned. should be fine-tuned. Herein, as a part of our ongoing investigations of disaccharide chemistry [13–16], we describe an efficient synthetic method that allowed us to obtain sambubiose in a satisfactory and higher overall

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 12 yield than previously reported [10] as well as to achieve its full characterization. The proposed methodology ensured an effective stereospecific glycosylation control and exploited strategies of protection and deprotection of functional groups which also guide the regioselective reaction progress.

2. Results and Discussion PharmaceuticalsThe study2020 ,started13, 198 with the optimization of the key step, consisting of the O-glycosylation3 of 13 reaction between the appropriately protected glucose (acceptor) and protected/activated xylose Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 12 (donor)Herein, (Scheme as a part1, step of “v”). our ongoing In particular, investigations our attention of disaccharide was first focused chemistry on the [13– synthesis16], we describe of several an glucose acceptors and xylose donors to overcome the abovementioned problems [10]. With regard to yieldefficient than synthetic previously method reported that allowed [10] as uswell to obtainas to achieve sambubiose its full in acharacterization. satisfactory and The higher proposed overall the glucose acceptor, the synthesis of compounds 9–12 (Figure 2) was designed and explored aiming yieldmethodology than previously ensured reportedan effective [10 ]stereospecific as well as to gl achieveycosylation its full control characterization. and exploited The strategies proposed of to avoid the acetylation and deacetylation steps necessary for the purification of 4a (Scheme 1). protectionmethodology and ensured deprotection an effective of functional stereospecific groups glycosylation which also controlguide andthe regioselective exploited strategies reaction of protectionprogress. and deprotection of functional groups which also guide the regioselective reaction progress. 2. Results and Discussion 2. Results and Discussion The study started with the optimization of the key step, consisting of the O-glycosylation The study started with the optimization of the key step, consisting of the O-glycosylation reaction between the appropriately protected glucose (acceptor) and protected/activated xylose (donor) reaction between the appropriately protected glucose (acceptor) and protected/activated xylose (Scheme1, step “v”). In particular, our attention was first focused on the synthesis of several glucose (donor) (Scheme 1, step “v”). In particular, our attention was first focused on the synthesis of several acceptors and xylose donors to overcome the abovementioned problems [10]. With regard to the glucose acceptors and xylose donors to overcome the abovementioned problems [10]. With regard to glucose acceptor, the synthesis of compounds 9–12 (Figure2) was designed and explored aiming to the glucose acceptor, the synthesis of compounds 9–12 (Figure 2) was designed and explored aiming avoid the acetylationFigure and deacetylation 2. Chemical structure steps necessary of synthesized for the glycoside purification acceptors. of 4a (Scheme1). to avoid the acetylation and deacetylation steps necessary for the purification of 4a (Scheme 1).

Methyl 3,5,6-tri-O-benzyl-D-glucofuranoside (9) is very easy to purify and relatively stable under basic or acid conditions. Compound 9 was synthesized starting from 3,5,6-tri-O-benzyl-1,2-O- isopropylidene-D-glucofuranoside (2, Scheme 2) (1% HCl, MeOH, 65 °C, 1.5 h, 91%; α/β 1:1.3), previously obtained by 1,2-O-isopropylidene-D-glucofuranoside (1, Scheme 2). Likewise, tert-butyldimethylsilyl 3,5,6-tri-O-benzyl-D-glucofuranoside (10, Figure 2) was synthesized from 2 via a vicinal diol intermediate. Unfortunately, 10 was obtained with a very low overall yield (1,4-dioxane, H2SO4, reflux, 3 h, 41%; TBSCl, imidazole, THF, 0 °C, 10 h, 10%; α/β 1:1). On the other hand, the attempt to change the “armed” protective groups with the “disarmed” tert- butyldimethylsilyl 3,5,6-tri-Figure O2. -acetyl-ChemicalD-glucofuranoside structure of synthesized (11, Figure glycoside 2) also acceptors. proved not to be effective in term of yields (Ac2O, Py, rt, 17 h, 85%; TFA, 50 °C, 3 h, 19%; TBSCl, imidazole, THF, 0 °C, 10 h; 10%).Methyl Both 103,5,6-tri- 3,5,6-tri- and 11OO-benzyl- -benzyl-D-glucofuranosidewere notD-glucofuranoside assessed as acceptors (9) ( 9is) very is in very easythe easy glycosylationto purify to purify and relativelyreaction. and relatively stableFinally, under stable our basicunderattention or basic acidwas or turnedconditions. acid conditions.to the Compound glycoside Compound acceptor 9 was allyl9synthesizedwas 3,5,6-tri- synthesized Ostarting-benzyl- starting fromD-glucofuranoside 3,5,6-tri- from 3,5,6-tri-O-benzyl-1,2- (12O-benzyl-, FigureO- 1,2-isopropylidene-2) whichO-isopropylidene-D-glucofuranoside was preparedD-glucofuranoside in an acidic ( 2environment, Scheme (2, Scheme 2) by(1%2 )treating (1%HCl, HCl, MeOH,2 with MeOH, allyl65 65°C, alcohol◦ C,1.5 1.5 h, [17] h, 91%; 91%; giving αα//ββ an1:1.3),1:1.3), α/β previously1:1.1 mixture obtained (Scheme by 2). 1,2- O-isopropylidene-D-glucofuranoside-isopropylidene-D-glucofuranoside (1, Scheme2 2).). Likewise, tert-butyldimethylsilyl 3,5,6-tri-O-benzyl-D-glucofuranoside (10, Figure 2) was synthesized from 2 via a vicinal diol intermediate. Unfortunately, 10 was obtained with a very low overall yield (1,4-dioxane, H2SO4, reflux, 3 h, 41%; TBSCl, imidazole, THF, 0 °C, 10 h, 10%; α/β 1:1). On the other hand, the attempt to change the “armed” protective groups with the “disarmed” tert- butyldimethylsilyl 3,5,6-tri-O-acetyl-D-glucofuranoside (11, Figure 2) also proved not to be effective in term of yields (Ac2O, Py, rt, 17 h, 85%; TFA, 50 °C, 3 h, 19%; TBSCl, imidazole, THF, 0 °C, 10 h; 10%). Both 10 and 11 were not assessed as acceptors in the glycosylation reaction. Finally, our Scheme 2. Reagents and conditions: (i) 80% NaH,NaH, BnBr,BnBr, DMF,DMF, rt, rt, 6 6 h, h, 70%; 70%; (ii) (ii) AllOH, AllOH, TsOH, TsOH, reflux, reflux, 3 attention was turned to the glycoside acceptor allyl 3,5,6-tri-O-benzyl-D-glucofuranoside (12, Figure h,3 h, 85%. 85%. 2) which was prepared in an acidic environment by treating 2 with allyl alcohol [17] giving an α/β 1:1.1 Likewise,Withmixture regard (Schemetert to-butyldimethylsilyl the 2). donor glycoside, 3,5,6-tri- only compoundsO-benzyl-D-glucofuranoside that are more stable ( 10than, Figure 5a (Scheme2) was 1), synthesizedsuch as fluoride from [18]2 via and a vicinaltrichloroacetimidate diol intermediate. [19] derivatives, Unfortunately, two10 ofwas the obtainedmost appealing with a veryand lowpromising overall glycoside yield (1,4-dioxane, donors for H O2SO-glycosylation,4, reflux, 3 h, were 41%; taken TBSCl, into imidazole, account. THF,Hence, 0 ◦ 2,3,4-tri-C, 10 h,O 10%;-acetyl-α/β 1:1).α-D-xylopyranosyl On the other hand, fluoride the attempt (13, to changeScheme the “armed”3) and protective2,3,4-tri- groupsO-acetyl- withα-D the-xylopyranosyl “disarmed” terttrichloroacetimidate-butyldimethylsilyl (14a 3,5,6-tri-, SchemeO-acetyl-D-glucofuranoside 3) were synthesized. (11, Figure2) also proved not to be e ffective in termIn ofthe yields case (Ac of2 O,fluoride Py, rt, 1713 h, [20], 85%; TFA,the desired 50 ◦C, 3 h,hemiacetal 19%; TBSCl, intermediate imidazole, THF,2,3,4-tri- 0 ◦C,O 10-acetyl- h; 10%).D- Bothxylopyranoside10 and 11 were (17, notScheme assessed 3) aswas acceptors prepared in thestarting glycosylation from D-(+)-xylose reaction. Finally, (15) through our attention an initial was turned Scheme to the 2. glycoside Reagents and acceptor conditions: allyl 3,5,6-tri-(i) 80% NaH,O-benzyl-D-glucofuranoside BnBr, DMF, rt, 6 h, 70%; (ii) (AllOH,12, Figure TsOH,2) whichreflux, was prepared3 h, 85%. in an acidic environment by treating 2 with allyl alcohol [17] giving an α/β 1:1.1 mixture (Scheme2). With regard regard to to the the donor donor glycoside, glycoside, only compounds only compounds that are thatmore are stable more than stable 5a (Scheme than 1),5a (Schemesuch as 1fluoride), such [18] as fluoride and trichloroacetimidate [ 18] and trichloroacetimidate [19] derivatives, [19 two] derivatives, of the most two appealing of the mostand promisingappealing andglycoside promising donors glycoside for O-glycosylation, donors for Owere-glycosylation, taken into account. were taken Hence, into 2,3,4-tri- account.O-acetyl- Hence, α-D-xylopyranosyl fluoride (13, Scheme 3) and 2,3,4-tri-O-acetyl-α-D-xylopyranosyl trichloroacetimidate (14a, Scheme 3) were synthesized. In the case of fluoride 13 [20], the desired hemiacetal intermediate 2,3,4-tri-O-acetyl-D- xylopyranoside (17, Scheme 3) was prepared starting from D-(+)-xylose (15) through an initial

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Pharmaceuticalsacetylation (Ac20202,O,13, 198Py, 0 °C, 6 h, 99%) followed by monohydrolysis of the corresponding4 of D 13- xylopyranose 1,2,3,4-tetracetate (16a) [21] with ammonium acetate (DMF dry, rt, 22 h, 70%). Glycoside donor 13 was then obtained by fluorination of 17 with diethylaminosulfur trifluoride (DAST)2,3,4-tri- O(CH-acetyl-2Cl2, α−-D-xylopyranosyl30 °C to rt, 1 h, 98%) fluoride as a ( 13mixture, Scheme of3 )anomers and 2,3,4-tri- (α/β O1:4),-acetyl- whichα-D-xylopyranosyl could be easily isolatedtrichloroacetimidate by a flash chromatography. (14a, Scheme3) were synthesized.

Scheme 3. ReagentsReagents and conditions: ( ii)) Ac Ac2O,O, Py, Py, 0 °C,◦C, 6 6 h, h, 75%; 75%; ( (iiii)) NH NH44OAc,OAc, DMF, DMF, rt, rt, 22 22 h, h, 70%; 70%; ( iiiiii)) DAST, CH22Cl2,, −3030 °C◦C to to rt, rt, 1 1 h, h, 98%; 98%; (iv (iv) )CCl CCl3CN,3CN, CH CH2Cl2Cl2, 20, 0°C,◦C, DBU, DBU, 1 h, 1 h,60%. 60%. −

WithIn the regard case to of trichloroacetimidate fluoride 13 [20], 14a the [22], desired this donor hemiacetal type showed intermediate some advantages 2,3,4-tri-O such-acetyl- as aD-xylopyranoside relatively high stability, (17, Scheme easy purification,3) was prepared and, most starting importantly, from D-( a+ potential)-xylose (stereochemical15) through an control initial ofacetylation the glycosylation (Ac2O, Py, 0 ◦C,re 6action. h, 99%) In followed addition, by monohydrolysis 14a was synthesized of the corresponding by treating D-xylopyranose 17 with 1,2,3,4-tetracetatetrichloroacetonitrile (16a (CCl)[213]CN) with and ammonium 1,8-diazabicyclo acetate (DMF [5.4.0]undec-7-ene dry, rt, 22 h, 70%). (DBU) Glycoside obtaining donor only13 was α- then obtained by fluorination of 17 with diethylaminosulfur trifluoride (DAST) (CH Cl , 30 C to rt, anomer [23]. As previously reported, DBU promoted thermodynamic control of the2 reaction,2 − ◦ which 1led h, to 98%) a more as a mixturestable α of-anomer anomers 14a (α (Scheme/β 1:4), which 3). One-pot could be attempts easily isolated to obtain by 14a a flash from chromatography. 16a were also explored;With however, regard to trichloroacetimidatesuch attempts proved14a not[22 to], be this advantageous donor type showed due to somethe fact advantages of their low such yields as a (datarelatively not shown). high stability, easy purification, and, most importantly, a potential stereochemical control of the glycosylationFinally, the key reaction. synthetic In addition, step, consisting14a was in synthesized the O-glycosylation by treating reaction,17 withtrichloroacetonitrile was explored. The (CClinitial3CN) substitution and 1,8-diazabicyclo of mercuric [5.4.0]undec-7-ene salts [10] with silver (DBU) oxide obtaining or silver only carbonateα-anomer as [23 an]. Asactivator previously was ineffectivereported, DBU any promotedadvantage thermodynamic because the yields control in these of the conditions reaction, fell which drastically led to a (data more not stable shown).α-anomer The 14ause (Schemeof safer, 3greener). One-pot catalysts attempts such to as obtain boron14a trifluoridefrom 16a diethylwere also etherate explored; (BF3 however,. OEt2) or suchtrimethylsilyl attempts provedtrifluoromethanesulfonate not to be advantageous (TMSOTf, due to particularly the fact of their suitable low for yields the (dataglycoside not shown). donor activation bearing the trichloroacetoimidateFinally, the key synthetic group step, as the consisting leaving group), in the O warrant-glycosylation further reaction,investigation was explored.as alternatives The toinitial mercury substitution compounds. of mercuric Dichloromethane salts [10] with was silver us oxideed as or a silvernon-polar carbonate solvent; as an therefore, activator wasthe stereoselectionineffective any advantageof glycosylation because was the yieldsled by inthe these 2-O conditions-acyl vicinal fell group drastically on the (data donor not shown).(anchimeric The . assistance).use of safer, All greener the prepared catalysts glycoside such as boron donors trifluoride and acceptors diethyl were etherate reacted (BF 3in OEtdifferent2) or trimethylsilylconditions in thetrifluoromethanesulfonate presence of powdered (TMSOTf,molecular particularlysieves (MS) suitable4Å (Scheme for the 4) at glycoside −20 °C, donorand the activation main results bearing are reportedthe trichloroacetoimidate in Table 1. group as the leaving group), warrant further investigation as alternatives to mercury compounds. Dichloromethane was used as a non-polar solvent; therefore, the stereoselection of glycosylation was led by the 2-O-acyl vicinal group on the donor (anchimeric assistance). All the prepared glycoside donors and acceptors were reacted in different conditions in the presence of powdered molecular sieves (MS) 4Å (Scheme4) at 20 C, and the main results are reported in Table1. − ◦ Firstly, the reactivity of fluoride glycoside 13b was explored with the methoxy derivative 9b or 9a as a glycoside acceptor, in the presence of BF . OEt at 20 C to room temperature for 4 h. 3 2 − ◦ The reaction gave the corresponding β- or α-disaccharide in 80% or 50% yields, respectively, and a complete trans stereocontrol (entries 1 and 2). A complete recovery of the acceptor (9b or 9a) was pointed out in both reactions. The lower yield for glycosylation obtained using the acetate 9a could be attributed to the steric hindrance of the methoxy group present in the same part of the plane of the sugar of the reactive 2-OH group. Similarly, the allyloxy acceptor 12b gave the corresponding coupling product in 30% yield, and its anomer 12a did not react at all and was completely recovered (entries 6 and 3, respectively). The absence of reactivity of 12a could be explained by the greater steric hindrance of its 1-allyloxy substituent compared to Scheme 4. General scheme of O-glycosylation. that of 1-methoxy present on 9a (entry 3 versus 2). On the other hand, the α-anomer fluoride donor 13a did not react with either highly reactive acceptor 9b or 12b in the same conditions (entries 4 and

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acetylation (Ac2O, Py, 0 °C, 6 h, 99%) followed by monohydrolysis of the corresponding D- xylopyranose 1,2,3,4-tetracetate (16a) [21] with ammonium acetate (DMF dry, rt, 22 h, 70%). Glycoside donor 13 was then obtained by fluorination of 17 with diethylaminosulfur trifluoride (DAST) (CH2Cl2, −30 °C to rt, 1 h, 98%) as a mixture of anomers (α/β 1:4), which could be easily isolated by a flash chromatography.

Scheme 3. Reagents and conditions: (i) Ac2O, Py, 0 °C, 6 h, 75%; (ii) NH4OAc, DMF, rt, 22 h, 70%; (iii) DAST, CH2Cl2, −30 °C to rt, 1 h, 98%; (iv) CCl3CN, CH2Cl2, 0 °C, DBU, 1 h, 60%.

With regard to trichloroacetimidate 14a [22], this donor type showed some advantages such as a relatively high stability, easy purification, and, most importantly, a potential stereochemical control of the glycosylation reaction. In addition, 14a was synthesized by treating 17 with trichloroacetonitrile (CCl3CN) and 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) obtaining only α- anomer [23]. As previously reported, DBU promoted thermodynamic control of the reaction, which led to a more stable α-anomer 14a (Scheme 3). One-pot attempts to obtain 14a from 16a were also explored; however, such attempts proved not to be advantageous due to the fact of their low yields Pharmaceuticals 2020, 13, 198 5 of 13 (data not shown). Finally, the key synthetic step, consisting in the O-glycosylation reaction, was explored. The 5, respectively).initial substitution The of last mercuric donor tosalts be investigated[10] with silver was oxide trichloroacetimidate or silver carbonate14a as, whichan activator was reacted was ineffective any advantage because the yields. in these conditions fell drastically (data not shown). The with 9b (entry 7) in the presence of BF3 OEt2, giving the corresponding β-linked disaccharide in . 44%use yield of safer, or withgreener12b catalysts(entry 8)such using as boron TMSOTf trifluoride as a promoter diethyl etherate and obtaining (BF3 OEt a2) betteror trimethylsilyl yield (60%), completetrifluoromethanesulfonate stereocontrol, and (TMSOTf, high reproducibility. particularly Surprisingly,suitable for the the glycoside use of the donor latter activation conditions bearing (donor 14atheand trichloroacetoimidate promoter TMSOTf) group and veryas the low leaving reactive group), donor warrant12a allowed further investigation coupling with as thealternatives formation to mercury compounds. Dichloromethane was used as a non-polar solvent; therefore, the of the corresponding disaccharide; however, with a lower yield (45%) and longer reaction time than stereoselection of glycosylation was led by the 2-O-acyl vicinal group on the donor (anchimeric anomer 12b (entry 9). Hence, with regard to O-glycosylation, the best overall result in terms of yield, assistance). All the prepared glycoside donors and acceptors were reacted in different conditions in stereocontrol, and reproducibility was obtained by reacting donor 14a and acceptor 12b promoted the presence of powdered molecular sieves (MS) 4Å (Scheme 4) at −20 °C, and the main results are by TMSOTf. reported in Table 1.

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 13 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 13 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 5 of 13 PharmaceuticalsFirstly, the 20reactivity20, 13, x FOR of PEERfluoride REVIEW glycoside 13b was explored with the methoxy derivative 9b or5 of 13 9a asPharmaceuticals a glycosideFirstly, 20the 20acceptor, ,reactivity 13, x FOR in PEER ofthe fluoride REVIEWpresence glycoside of BF3 . 13bOEt 2was at − explored20 °C to roomwith thetemperature methoxy derivativefor 4 h. The 59b of or13 PharmaceuticalsPharmaceuticalsFirstly, the20 2020 reactivity,20 13, ,1 x3 ,FOR x FOR PEER of PEER fluoride REVIEW REVIEW glycoside 13b was. explored with the methoxy derivative 9b5 of5 or13of 13 reaction9a as Firstly, gavea glycoside the the correspondingreactivity acceptor, of in fluoride the β- orpresence αglycoside-disaccharide of BF 13b3 OEtwas in 2 80%exploredat − 20 or °C 50% with to room yields, the methoxytemperature respectively, derivative for and 4 h. 9b aThe or 9a as a glycoside acceptor, in the presence of BF3 . OEt2 at −20 °C to room temperature for 4 h. The completereactionFirstly, trans gave stereocontrol the the reactivity corresponding (entries of fluoride 1 βand- glycosideor 2). α -disaccharide 13b. was explored in 80% or with 50% the yields, methoxy respectively, derivative and9b or a reaction9a Firstly,asFirstly, a gaveglycoside the the the reactivity reactivity corresponding acceptor, of offluoride in fluoride the β -presence glycosideor glycoside α-disaccharide of 13b BF 13b3 was OEtwas explored in2 explored at 80% −20 or °Cwith 50%withto theroom yields,the methoxy methoxytemperature respectively, derivative derivative for 4 and9b h. 9b orThe a or 9acomplete as a glycoside trans stereocontrol acceptor, in (entries the presence 1 and 2).of BF3 . OEt2 at −20 °C to room temperature for 4 h. The complete9areaction9a as as a glycosidea transglycoside gave stereocontrol the acceptor, acceptor, corresponding in (entries inthe the presence presence β1 -and or α2). of- disaccharide ofBF BF3 . 3OEt . OEt2 at2 inat−20 − 80%20 °C °C to or to room 50% room yields,temperature temperature respectively, for for 4 h.4 h. andThe The a reaction gave the correspondingTable 1. Optimization β- or α- ofdisaccharide the O-glycosylation in 80% reaction or 50%. yields, respectively, and a reactioncompletereaction gave gavetrans the thestereocontrol corresponding corresponding (entries β - βor- 1or andα -αdisaccharide- 2).disaccharide in in 80% 80% or or 50% 50% yields, yields, respectively, respectively, and and a a complete trans stereocontrolTable (entries 1. Optimization 1 and 2). of the O-glycosylation reaction. completecomplete trans trans stereocontrol stereocontrolTable (entries (entries 1. Optimization 1 and1 and 2). 2). of the O-glycosylationReaction reaction. Yield Entry Donor SchemeSchemeAcceptor 4.4. GeneralGeneral scheme Catalyst of of OO-glycosylation.-glycosylation. Product Table 1. Optimization of the O-glycosylationTime reactionReaction (h) . (%)Yield Entry Donor Table 1. OptimizationAcceptor of the O-glycosylationCatalyst Reaction reaction . Product Yield Entry DonorTable TableTable 1. 1.Optimization Optimization1. OptimizationAcceptor of ofthe the the OO -OCatalyst-glycosylationglycosylation-glycosylation reaction reaction.ReactionreactionTime. (h). Product Yield(%) Entry Donor Acceptor Catalyst Time (h) Product (%) ReactionTime (h) Yield(%) 1Entry Donor Acceptor BF3 Catalyst. OEt2 ReactionReaction4 Reaction 18bProduct Yield80Yield EntryEntryEntry Donor DonorDonor AcceptorAcceptor Acceptor CatalystCatalyst Catalyst Time (h) ProductProductProduct (%) Yield (%) 1 BF3 . OEt2 4 18b 80 TimeTimeTime (h) (h) (h) (%)(%) 1 BF3 . OEt2 4 18b 80 . 1 BF3 OEt2 4 18b 80 3 . 2 1 BF OEt. 4 18b 80 1 1 1 BFBF3 BF. OEt3 .3 OEtOEt2 2 2 4 4 4 18b18b18b 8080 80 2 13b BF3 . OEt2 4 18a 50

2 13b BF3 . OEt2 4 18a 50

2 13b BF3 . OEt2 4 18a 50 3 . 2 2 13b BF OEt. 4 18a 50 2 2 13b13b BFBF3 .3 OEtOEt2 2 4 4 18a18a 50 50 2 2 13b13b BFBF3 . OEt3 . OEt2 2 4 4 18a18a 5050 3 13b BF 3 . OEt2 6 19a - 3 13b BF3 . OEt2 6 19a -

3 13b BF3 . OEt2 6 19a - . 3 3 13b13b BFBF3 .3 OEtOEt2 2 6 6 19a19a - - 3 13b BF3 . OEt2 6 19a - . . 4 3 3 13b13b 9b BF BF3 BF.3 OEt OEt3 OEt2 2 2 6 6 6 18b19a 19a - - -

4 9b BF3 . OEt2 6 18b - . . 4 4 9b9b BF3 BFOEt3 2OEt 2 6 6 18b18b - - . 4 9b BF3 OEt2 6 18b - 4 9b BF3 . OEt2 6 18b - . 4 9b BF.3 OEt3 . 2 2 6 18b - 5 4 13a 9b BF3 BFOEt OEt2 6 6 19b18b - - . 5 5 13a13a BFBF3 . OEtOEt2 6 6 19b19b - - 3 2 5 13a BF3 . OEt2 6 19b - . 5 13a BF. 3 OEt2 6 19b - 6 13b 12b BF3 OEt. 2. 6 19b 30 6 5 13b13a 12b BFBF3 3. OEtOEt2 2 6 6 19b19b - 30 5 56 13a13b13a 12b BFBF3 . OEt3 . OEt2 2 6 6 19b19b - 30- 6 13b 12b BF3 . OEt2 6 19b 30 3 . 2 6 13b 12b BF OEt. 6 19b 30 7 7 6 13b 9b 12b9b BF3 BF. OEtBF3 .3 OEt2 OEt2 2 12 6 12 18b19b 18b 44 30 44 . . 6 67 13b13b 12b12b9b BFBF3 OEt3 . OEt2 2 6 126 19b19b18b 303044 7 9b BF3 . OEt2 12 18b 44 8 7 14a 12b9b BFTMSOTf3 . OEt2 12 3.5 18b19b 44 60 . 9 8 7 14a14a 12b 12a9b TMSOTfBFTMSOTf3 OEt 2 3.5 12 6 19b18b 19a 60 44 45 7 7 9b9b BFBF3 . OEt3 . OEt2 2 1212 18b18b 4444 9 8 14a 14a 12a 12b TMSOTfTMSOTf 6 3.5 19a 19b 45 60 8 9 14a14a 12b12a TMSOTfTMSOTf 3.5 6 19b19a 6045 9 8 14a14a 12a12b TMSOTfTMSOTf 6 3.5 19a19b 4560 A complete8 recovery14a of the acceptor (9b12b or 9a) was pointedTMSOTf out in both3.5 reactions . The19b lower 60yield 8 98 14a14a 12b12b12a TMSOTfTMSOTf 3.53.5 6 19b19b19a 604560 for glycosylationA9 complete obtained recovery14a using of the the acceptor acetate12a (9b 9a or could 9a) was beTMSOTf pointed attributed out to in theboth6 steric reactions hindrance19a. The lower of45 the yield A 9complete 9 recovery14a14a of the acceptor12a (9b12a or 9a) wasTMSOTf pointedTMSOTf out in both6 6 reactions19a19a. The lower4545 yield methoxyfor glycosylation group present obtained in the using same the part acetate of the 9a plane could of be the attributed sugar of to the the reactive steric hindrance2-OH group of. the for glycosylationA complete obtained recovery usingof the acceptor the acetate (9b 9a or 9acould) was be pointed attributed out in to both the reactions steric hindrance. The lower of theyield Similarly,methoxyA completethe group allyloxy recovery present acceptor inof the 12b acceptor samegave the part (9b corresponding of or the9a) was plane pointed ofcoupling the out sugar inproduct both of thereactions in reactive30% yield. The 2- ,lOH owerand group its yield . methoxyforA glycosylation Acomplete complete group recovery present recovery obtained inof of the using the acceptor same acceptor the part acetate(9b ( of9b or theor9a 9a 9a) was planecould) was pointed pointedof be the attributed out sugar out in inboth of both to the reactions the reactions reactive steric. The hindrance. The2- OHlower lower group yield of yield the. anomerforSimilarly, glycosylation 12a did the not allyloxy reactobtained acceptorat all using and 12b was the gave acetatecompletely the corresponding9a could recovered be attributed coupling(entries 6 to productand the 3 steric, respectively in 30% hindrance yield)., The and of the its Similarly,formethoxyfor glycosylation glycosylation the group allyloxy obtained present obtained acceptor inusing using the 12b thesame thegave acetate acetatepart the ofcorresponding9a 9a thecould could plane be be attributedof coupling attributed the sugar toproduct to theof the the steric stericin reactive 30% hindrance hindrance yield 2-OH, and of group of theits the . absencemethoxyanomer of reactivity12a group did presentnot of react12a in couldat the all sameand be explained was part completely of theby planethe recovered greater of the steric sugar(entries hindrance of 6 the and reactive 3 of, respectively its 12-allyloxyOH group). The . anomermethoxySimilarly,methoxy 12a group groupthedid allyloxynot present present react acceptor in at inthe all the sameand 12b same was gave part part completely ofthe of the corresponding the plane plane recovered of of the thecoupling sugar(en sugartries of product 6of theand the reactive 3 reactive , inrespectively 30% 2- OHyield2-OH group,). and groupThe .its . substituentSimilarly,absence compared of the reactivity allyloxy to that ofacceptor 12aof 1 -couldmethoxy 12b gave be present explained the corresponding on 9aby (entrythe greater 3 coupling versus steric 2) .product On hindrance the otherin 30% ofhand, yieldits 1the-,allyloxy and α- its absenceSimilarly,anomerSimilarly, of the12a reactivitythe allyloxydid allyloxy not ofacceptorreact acceptor12a atcould all 12b 12band begave gavewas explained the thecompletely corresponding corresponding by the recovered greater coupling coupling steric(entries product hindranceproduct 6 and in in330% , of30%respectively itsyield yield 1-,allyloxy and, and). itsThe its anomeranomersubstituent fluoride 12a compareddid donor not 13areact to didthat at not allof 1reactand-methoxy was with completely presenteither highly on recovered9a (entryreactive 3 (en versusacceptortries 26) . 9bandOn or the3 ,12b respectivelyother in thehand, same ).the The α- substituentanomerabsenceanomer 12a 12a of compared did reactivitydid not not react to react ofthat at12a atofall all1could and-methoxy and was bewas explainedcompletely present completely on by 9arecovered recoveredthe (entry greater 3 (enversus (en triessterictries 2 6) . hindranceOnand6 and the 3, 3respectivelyother, respectively of hand,its 1- allyloxythe). ).The αThe- conditionsabsenceanomer (entries fluoride of reactivity 4 donorand 5 of, 13a respectively12a did could not react be). The explained with last donoreither by highlytothe be greater investigated reactive steric acceptor was hindrance trichloroacetimidate 9b or of12b its in 1 -theallyloxy same anomerabsencesubstituentabsence fluoride of of reactivity compared reactivity donor of 13a toof12a that 12adid could of couldnot 1- methoxyreact be be explained with explained present either by byon highlythe 9athe greater(entry greaterreactive 3 steric versus stericacceptor hindrance 2 hindrance). On 9b theor of12bother ofits inits hand,1 the -allyloxy1-allyloxy same the α - 14asubstituent,conditions which was (entriescompared reacted 4 withand to that 59b, respectively of(entry 1-methoxy 7) in). theThe present presence last donoron 9a of (entry toBF be3 . investigatedOEt 3 versus2, giving 2). Onthewas the corresponding trichloroacetimidate other hand, theβ- α- conditionssubstituentanomersubstituent fluoride (entries compared compared donor4 and to to that 513a ,that respectively ofdid of1- methoxy1not-methoxy react). The presentwith present last either donor on on 9a highly 9a (entryto (entrybe investigatedreactive 3 versus3 versus acceptor 2) .2 On)was. On the trichloroacetimidate9b the other or other 12b hand, hand,in the the thesame α- α - anomer fluoride donor 13a did not react with either highly reactive3 . acceptor2 9b or 12b in the same linkedconditions14a disaccharide, which (entrieswas reactedin 444% and yieldwith 5, respectively or9b with(entry 12b 7)). The(entryin the last 8)presence donor using toTMSOTf of be BF investigated OEtas a ,promotergiving was the trichloroacetimidate and corresponding obtaining a β- 14aanomeranomer, which fluoride fluoride was reacted donor donor 13awith 13a did 9bdid not (entry not react react 7) within with the either presenceeither highly highly of reactiveBF reactive3 . OEt acceptor2 ,acceptor giving 9bthe 9b or corresponding or 12b 12b in inthe the same same β- betterconditionslinked yield disaccharide (60%) (entries, complete 4 inand 44% stereocontrol5, respectivelyyield or with, and). 12bThe high (entrylast reproducib donor 8) using to ilitybe TMSOTf investigated. Surprisingly,. as a promoter was the trichloroacetimidate use and of the obtaining latter a linkedconditions14aconditions, whichdisaccharide (entries (entrieswas reacted 4 in and4 44%and 5 with, yield5respectively, respectively 9b or (entry with). 12b 7)The). Thein (entry last the last donorpresence 8)donor using to tobe ofTMSOTf be investigatedBF investigated3 OEt as 2a, givingpromoter was was trichloroacetimidate the trichloroacetimidate andcorresponding obtaining a β - 14abetter, which yield was(60%) reacted, complete with stereocontrol 9b (entry 7), inand the high presence reproducib of BFility3 . OEt. Surprisingly,2, giving the the corresponding use of the latter β- conditionslinked disaccharide (donor 14a andin 44% promoter yield or TMSOTf) with 12b and (entry very 8) usinglow reactive TMSOTf. . donor as a promoter12a allowed and coupling obtaining a better14a14a, which, yieldwhich was(60%) was reacted, reactedcomplete with with stereocontrol 9b 9b (entry (entry 7) , 7) andin inthe highthe presence presence reproducib of ofBF ilityBF3 3OEt . Surprisingly,OEt2, giving2, giving the thethe corresponding correspondinguse of the latter β- β- withlinkedconditions the formation disaccharide (donor of the14a in corresponding 44%and yieldpromoter or with disaccharide TMSOTf) 12b (entry and; however 8) very using low, TMSOTfwith reactive a lower as donor a yieldpromoter 12a (45%) allowed and and obtaining long couplinger a conditionslinkedbetterlinked disaccharide yield disaccharide (donor (60%) 14a, incomplete in44%and 44% yieldpromoter yield stereocontrol or or with TMSOTf)with 12b 12b, and (entry (entryand high 8)very reproducib 8)using using low TMSOTf reactiveTMSOTfility. Surprisingly,as donor as a promotera promoter 12a allowed the and andus obtaininge ofobtainingcoupling the latter a a reactionbetterwith timethe yield formation than (60%) anomer, complete of the 12b corresponding (entry stereocontrol 9). Hence disaccharide, and, with high regard reproducib; however to O-glycosylation,ility, with. Surprisingly, a lower the yield best the (45%)overall use of and resultthe long latter er withbetterconditionsbetter the yield yieldformation (60%) (donor (60%), complete of, complete14a the andcorresponding stereocontrol promoterstereocontrol TMSOTf) disaccharide, and, and high high and reproducib ;reproducib howeververy lowility, withreactiveility. Surprisingly,. aSurprisingly, lower donor yield 12a the (45%)the allowed us use ofande ofthe couplingthelong latter latterer in termsconditionsreaction of yield time (donor, thanstereocontrol anomer14a and 12b , promoterand (entry reproducibility 9).TMSOTf) Hence, withwasand obtainedregardvery low to byOreactive- glycosylation,reacting donor donor 12a the 14a allowedbest and overall acceptor coupling result reactionconditionswithconditions the time formation(donor than(donor anomer14a 14aof and the and 12b correspondingpromoter promoter (entry 9). TMSOTf) HenceTMSOTf) disaccharide, with and and regardvery very; however low tolow Oreactive- glycosylation,reactive, with donora lowerdonor 12a the yield 12a allowedbest allowed (45%) overall couplingand coupling result long er 12bwithin promoted terms the formationof byyield TMSOTf., stereocontrol of the corresponding, and reproducibility disaccharide was; however obtained, with by reacting a lower donoryield (45%) 14a and and acceptor longer withreactionwith the the formation timeformation than of anomer ofthe the corresponding corresponding 12b (entry 9). disaccharide Hence disaccharide, with; howeverregard; however to, Owith,- withglycosylation, a lowera lower yield yield the (45%) best (45%) overalland and long long resulter er inreaction 12bterms promoted of time yield than, bystereocontrol TMSOTf.anomer 12b , and (entry reproducibility 9). Hence, with was regard obtained to O by-glycosylation, reacting donor the 14a best and overall acceptor result reactioninreaction terms time timeof yieldthan than ,anomer stereocontrol anomer 12b 12b (entry ,(entry and 9). reproducibility 9). Hence Hence, with, with regard was regard obtained to toO -Oglycosylation,-glycosylation, by reacting donorthe the best best 14a overall overalland acceptor result result 12bin promotedterms of yield by TMSOTf., stereocontrol , and reproducibility was obtained by reacting donor 14a and acceptor in 12bin terms terms promoted of ofyield yield ,by stereocontrol, stereocontrolTMSOTf. , and, and reproducibility reproducibility was was obtained obtained by by reacting reacting donor donor 14a 14a and and acceptor acceptor 12b promoted by TMSOTf. 12b 12b promoted promoted by by TMSOTf. TMSOTf.

Pharmaceuticals 2020, 13, 198 6 of 13

We then focused on the deprotection steps of 3,5,6-tri-O-benzyl-2-O-(2,3,4-tri-O-acetyl-β- D-xylopiranosyl)-D-glucofuranoside 18a and 18b or 19a and 19b (Scheme4) with the aim of maximizing the efficiency of the synthetic strategy to obtain sambubiose. O-Methoxy group deprotection in the disaccharides 18a and 18b were first investigated using strong acids such as hydrochloride acid (HCl, rt or 15 C, 10 h) or BF . OEt (0 C, 10 h). Unfortunately, − ◦ 3 2 ◦ demethylation was not obtained in any of these cases, and degradation of the starting material or no . reaction was observed. Also treating disaccharides with trityl tetrafluoroborate [(C6H5)3C BF4][24] (rt or 15 C, 4–12 h), usually employed in the deprotection of benzyl and methyl ethers on anomeric − ◦ carbons, was found to be detrimental (data not shown). Hence, 18b was also first debenzylated by hydrogenolysis (10% Pd/C, EtOAc/EtOH 1:2, 32%) leading to the derivative β-methyl 2-O-(2,3,4-tri-O-acetyl-β-D-xylopyranosyl)-D-glucofuranoside (20b) which, in turn, was deacetylated (NaOMe, MeOH, 7 h, 70%) to give β-methyl 2-O-β-D-xylopyranosyl-D-glucofuranoside (21b). Attempts to obtain sambubiose by deprotection of 20b and 21b by cleaving the methoxy group also failed (data not shown). In the case of allyloxy disaccharides 19a and 19b, sambubiose might theoretically be obtained by two different deprotection routes (compound 19b is reported in Scheme5 as an example), both of which were explored. The first, on the left in Scheme5, envisaged O-deacetylation, O-deallylation, and, finally, O-debenzylation. On the other hand, the second route called for O-deallylation before O-deacetylation. The initial deprotection route involved O-deacetylation of compounds 19a or 19b. The compounds were treated using a typical condition for the hydrolysis of the acetyl esters with sodium methoxide and gave the corresponding desired compounds α- and β-allyl 3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-D-glucofuranoside in good yield (22a, 70%; 22b, 90%). Subsequently, the deprotection of the allyl group was investigated with 22a and 22b. Several methods were explored, including palladium chloride [25] (MeCOONa, MeCOOH, PdCl2,H2O, rt, 12 h, 42%), Wilkinson’s catalyst [26] [(Ph3P)3RhCl, toluene:MeOH, rt to reflux, 20 h, 39%] or hydrogen activated iridium complex [27] {[Ir(COD)(PCH3Ph2)2]PF6,H2, TsOH, CH2Cl2/MeOH, 19%}, but only β-anomer 22b reacted, probably due to the steric hindrance of α-one. Therefore, 22b was the only possible anomer suitable for the synthesis of 23 (3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-α-D-glucofuranose and 3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-β-D-glucofuranose) which was obtained as an anomeric mixture. On the other hand, using the second approach (on the right in Scheme5), 19a and 19b were deallylated with palladium chloride or Wilkinson’s catalyst under the previously described conditions. Disaccharide 19b gave the corresponding product 3,5,6-tri-O-benzyl-2-O- (2,3,4-tri-O-acetyl-β-D-xylopiranosyl)-D-glucofuranose (24) in good yields with both methods. However, compound 19a was deprotected only by (Ph3P)3RhCl) in low yield (23%). Then, 24 was deacetylated using sodium methoxide and methanol to give 23. In conclusion, the deprotection scheme on the left used to obtain 23 was more effective in terms of overall yield than the one on the right (38% versus 27%). Finally, sambubiose was obtained by O-debenzylation of 23 which was treated with palladium under hydrogen atmosphere. During the last deprotection, the five-membered ring (glucofuranose) of compound 23 spontaneously transformed into a six-membered ring (glucopyranose). Pharmaceuticals 2020, 13, x FOR PEER REVIEW 6 of 12

Unfortunately, demethylation was not obtained in any of these cases, and degradation of the starting material or no reaction was observed. Also treating disaccharides with trityl tetrafluoroborate [(C6H5)3C.BF4] [24] (rt or −15 °C, 4–12 h), usually employed in the deprotection of benzyl and methyl ethers on anomeric carbons, was found to be detrimental (data not shown). Hence, 18b was also first debenzylated by hydrogenolysis (10% Pd/C, EtOAc/EtOH 1:2, 32%) leading to the derivative β-methyl 2-O-(2,3,4-tri-O-acetyl-β-D-xylopyranosyl)-D-glucofuranoside (20b) which, in turn, was deacetylated (NaOMe, MeOH, 7 h, 70%) to give β-methyl 2-O-β-D- xylopyranosyl-D-glucofuranoside (21b). Attempts to obtain sambubiose by deprotection of 20b and 21b by cleaving the methoxy group also failed (data not shown). In the case of allyloxy disaccharides 19a and 19b, sambubiose might theoretically be obtained by two different deprotection routes (compound 19b is reported in Scheme 5 as an example), both of which were explored. The first, on the left in Scheme 5, envisaged O-deacetylation, O-deallylation, and, finally, O-debenzylation. On the other hand, the second route called for O-deallylation before O-deacetylation. The initial deprotection route involved O-deacetylation of compounds 19a or 19b. The compounds were treated using a typical condition for the hydrolysis of the acetyl esters with sodium methoxide and gave the corresponding desired compounds α- and β-allyl 3,5,6-tri-O-benzyl- 2-O-β-D-xylopyranosyl-D-glucofuranoside in good yield (22a, 70%; 22b, 90%). Subsequently, the deprotection of the allyl group was investigated with 22a and 22b. Several methods were explored, including palladium chloride [25] (MeCOONa, MeCOOH, PdCl2, H2O, rt, 12 h, 42%), Wilkinson’s catalyst [26] [(Ph3P)3RhCl, toluene:MeOH, rt to reflux, 20 h, 39%] or hydrogen activated iridium complex [27] {[Ir(COD)(PCH3Ph2)2]PF6, H2, TsOH, CH2Cl2/MeOH, 19%}, but only β-anomer 22b reacted, probably due to the steric hindrance of α-one. Therefore, 22b was the only possible anomer suitable for the synthesis of 23 (3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-α-D-glucofuranose and Pharmaceuticals3,5,6-tri-O-benzyl-2-2020, 13, 198O-β-D-xylopyranosyl-β-D-glucofuranose) which was obtained as an anomeric7 of 13 mixture.

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 7 of 12 acetyl-β-D-xylopiranosyl)-D-glucofuranose (24) in good yields with both methods. However, compound 19a was deprotected only by (Ph3P)3RhCl) in low yield (23%). Then, 24 was deacetylated using sodium methoxide and methanol to give 23. In conclusion, the deprotection scheme on the left used to obtain 23 was more effective in terms of overall yield than the one on the right (38% versus 27%).Scheme Finally, 5. sambubiose Reagents and was conditions: obtained ( i) byMeONa, MeONa, O-debenzylation MeOH, rt, 3 h, h, of 90% 90% 23 (which (22b) or or was 38% 38% treated( (2323);); ( (iiii)) withMeCOONa, MeCOONa, palladium underMeCOOH, hydrogen PdCl atmosphere.22,,H H2O,O, rt, rt, 12 12During h, h, 42% 42% the( (2323)) lastor or 70% 70% depr ( (2424otection,);); ( (iiiiii))H H22 ,, thePd/C Pd /five-memberedC 10%, 10%, EtOH, EtOH, rt, rt, 6 6ring h, h, 51%. 51%. (glucofuranose) of compound 23 spontaneously transformed into a six-membered ring (glucopyranose). TheOn thefinal final other four hand,improved/developed improved using/developed the second steps approach are reportedreported (on the inin SchemeSchemeright in6 6..Scheme 5), 19a and 19b were deallylated with palladium chloride or Wilkinson’s catalyst under the previously described conditions. Disaccharide 19b gave the corresponding product 3,5,6-tri-O-benzyl-2-O-(2,3,4-tri-O-

Scheme 6. ReagentsReagents and and conditions: ( (ii)) TMSOTf, TMSOTf, CH 2ClCl22, ,–20 –20 °C◦C to to rt, rt, 3.5 3.5 h, h, 60%; 60%; ( (iiii)) MeONa, MeONa, MeOH, MeOH, rt, 3 h, 90%; (iii) MeCOONa, MeCOOH, PdCl 2,,H H22O,O, rt, rt, 12 12 h, h, 42%; 42%; ( (iviv))H H22, ,Pd/C Pd/C 10%, 10%, EtOH, EtOH, rt, rt, 6 6 h, h, 51%.

Interestingly, sambubiose was obtained with a satisfactorysatisfactory overall yield (7%) starting from the acceptor precursor 1 compared to the 0.24% yield reported inin thethe literatureliterature [[10].10].

3. Materials and Methods 3. Materials and Methods Chemicals, Materials, and Methods Chemicals, Materials, and Methods All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA), Alfa Aesar (Haverhill, All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA), Alfa Aesar (Haverhill, MA, USA), or TCI (Tokyo, Japan) in the highest quality commercially available. The solvents were RP MA, USA), or TCI (Tokyo, Japan) in the highest quality commercially available. The solvents were grade. The melting points were determined using the Büchi (Flawil, Switzerland) B-540 apparatus. The RP grade. The melting points were determined using the Büchi (Flawil,1 Switzerland)13 B-540 apparatus.20 purity of the products was evaluated unequivocally through MS, H NMR, C NMR, IR, and [α] D. The purity of the products was evaluated unequivocally through MS, 1H NMR, 13C NMR, IR, and [α]20D. The MS (ESI) spectra were recorded with a Waters (Milford, MA, USA) Micromass ZQ spectrometer in a positive mode using a nebulizing nitrogen gas at 400 L/min and a temperature of 250 °C, cone flow 40 mL/min, capillary 3.5 K volts, and cone voltage 60 V; the revelation was performed either in ESI (+) and/or ESI (−), from 100 to 800 units of mass, spectrophotometrically measured using a diode array spectrophotometer. The 1H NMR and 13C NMR spectra were recorded on a Bruker (Billerica, MA, USA) AC 400 or 101 instrument and analyzed using the Top Spin 1.3 (2013) Bruker NMR software package. Chemical shifts were measured using the central peak of the solvent. Purification of the crude material was carried out by column chromatography on silica gel “flash” [230–400 mm, Merck (Darmstadt, Germany)]. The TLC analyses were performed on pre- coated aluminum oxide on aluminum sheets (60 F254, neutral; Merck). The IR spectra were obtained with a Thermo Fisher Scientific (Waltham, MA, USA) Nicolet Avatar 360 spectrophotometer. The optical rotations were measured on the Digital Perkin Elmer (Waltham, MA, USA) 241 polarimeter using a sodium lamp (589 nm) as a light source; concentrations were expressed in g/100 mL, and the length of the cell was 1 dm.

Synthesis of allyl 3,5,6-tri-O-benzyl-α-D-glucofuranoside (12a) and allyl 3,5,6-tri-O-benzyl-β-D- glucofuranoside (12b). To a solution of 2 (1 g, 2.04 mmol) [obtained by treating 1 (2 g, 9.08 mmol) with 80% NaH (0.9 g, 30 mmol), BnBr (4.66 g, 27.25 mmol) in DMF (40 mL)] at room temperature for 6 h in allyl alcohol (14.6 mL) and under stirring, TsOH (0.062 g, 0.33 mmol) was added. After the reactants

Pharmaceuticals 2020, 13, 198 8 of 13

The MS (ESI) spectra were recorded with a Waters (Milford, MA, USA) Micromass ZQ spectrometer in a positive mode using a nebulizing nitrogen gas at 400 L/min and a temperature of 250 ◦C, cone flow 40 mL/min, capillary 3.5 K volts, and cone voltage 60 V; the revelation was performed either in ESI (+) and/or ESI ( ), from 100 to 800 units of mass, spectrophotometrically measured using a diode array − spectrophotometer. The 1H NMR and 13C NMR spectra were recorded on a Bruker (Billerica, MA, USA) AC 400 or 101 instrument and analyzed using the Top Spin 1.3 (2013) Bruker NMR software package. Chemical shifts were measured using the central peak of the solvent. Purification of the crude material was carried out by column chromatography on silica gel “flash” [230–400 mm, Merck (Darmstadt, Germany)]. The TLC analyses were performed on pre-coated aluminum oxide on aluminum sheets (60 F254, neutral; Merck). The IR spectra were obtained with a Thermo Fisher Scientific (Waltham, MA, USA) Nicolet Avatar 360 spectrophotometer. The optical rotations were measured on the Digital Perkin Elmer (Waltham, MA, USA) 241 polarimeter using a sodium lamp (589 nm) as a light source; concentrations were expressed in g/100 mL, and the length of the cell was 1 dm.

Synthesis of allyl 3,5,6-tri-O-benzyl-α-D-glucofuranoside (12a) and allyl 3,5,6-tri-O-benzyl-β- D-glucofuranoside (12b). To a solution of 2 (1 g, 2.04 mmol) [obtained by treating 1 (2 g, 9.08 mmol) with 80% NaH (0.9 g, 30 mmol), BnBr (4.66 g, 27.25 mmol) in DMF (40 mL)] at room temperature for 6 h in allyl alcohol (14.6 mL) and under stirring, TsOH (0.062 g, 0.33 mmol) was added. After the reactants were refluxed for 3 h, the mixture was cooled, extracted with CH2Cl2, washed with NaHCO3 sat., dried over Na2SO4, filtered and concentrated. Purification of the residue by column chromatography (cyclohexane/EtOAc 9:1) gave 12a and 12b as light yellow oils, which were dried under N2 atmosphere because of their thermolability (Rf: 12a 0.4, 12b 0.3; cyclohexane/EtOAc 6:4). Yield: 12a 40% (0.4 g, + 1 0.82 mmol); 12b 45% (0.45 g, 0.92 mmol). 12a: MS (ESI): 490 (M + H ). H NMR (CDCl3) δ: 3.61 (dd, 6b 6a 1H, J6b–5 = 5.8 Hz, J6b–6a = 10.6 Hz, H ), 3.78 (dd, 1H, J6a–5 = 2.0 Hz, J6a–6b = 10.6 Hz, H ), 3.95 (ddd, 5 3 1H, J5–6a = 2.0 Hz, J5–6b = 5.8 Hz, J5–4 = 8.4 Hz, H ), 4.00 (dd, 1H, J3–2 = 1.9 Hz, J3–4 = 4.4 Hz, H ), 4.01 7b (dddd, 1H, J7b–9a = J7b–9b = 1.5 Hz, J7b–8 = 6.2 Hz, J7b–7a = 12.8 Hz, H OCHHCHCH2), 4.22 (dddd, 1H, 7a J7a–9a = J7a–9b = 1.5 Hz, J7a–8 = 5.2 Hz, J7a–7b = 12.8 Hz, H OCHHCHCH2), 4.26 (dd, 1H, J2–3 = 1.9 Hz, 2 4 J2–1 = 4.5 Hz, H ), 4.27 (dd, 1H, J4–3 = 4.4 Hz, J4–5 = 8.4 Hz, H ), 4.45 (d, 1H, J = 11.5 Hz, OCH2C6H5), 4.47 (d, 1H, J = 11.5 Hz, OCH2C6H5), 4.49 (d, 1H, J = 12.2 Hz, OCH2C6H5), 4.53 (d, 1H, J = 12.2 Hz, OCH2C6H5), 4.63 (d, 1H, J = 11.5 Hz, OCH2C6H5), 4.72 (d, 1H, J = 11.5 Hz, OCH2C6H5), 5.11 (d, 1H, 1 9b J1–2 = 4.5 Hz, H ), 5.12 (dddd, 1H, J9b–9a = J9b–7a = J9b–7b = 1.5 Hz, J9b–8 = 10.2 Hz, H OCH2CHCHH), 9a 5.19 (dddd, 1H, J9a–9b = J9a–7a = J9a–7b = 1.5 Hz, J9a–8 = 17.2 Hz, H OCH2CHCHH), 5.83 (dddd, 1H, 8 J8–7a = 5.2 Hz, J8–7b = 6.2 Hz, J8–b = 10.2 Hz, J8–9a = 17.2 Hz, H OCH2CHCH2), 7.24–7.37 (m, 15H, 13 ArH) ppm. C NMR (CDCl3) δ: 69.2, 71.2, 71.7, 72.6, 73.4, 76.1, 77.9, 83.9, 100.4, 117.7, 127.5, 127.5, 1 127.6, 128.1, 128.2, 128.3, 133.7, 137.9, 138.6, 138.9 ppm. IR (Nujol) ν = 3550, 1454, 1372, 1206, 1057 cm− . 20 + 1 [α] D = –56.8 (c 0.3, CHCl3). 12b: MS (ESI): 490 (M + H ). H NMR (CDCl3) δ: 3.63 (dd, 1H, J6b–5 = 6b 6a 5.3 Hz, J6b–6a = 10.7 Hz, H ), 3.80 (dd, 1H, J6a–5 = 2.0 Hz, J6a–6b = 10.7 Hz, H ), 3.90 (dddd, 1H, J7b–9a 7b = J7b–9b = 1.5 Hz, J7b–8 = 6.0 Hz, J7b–7a = 13.0 Hz, H OCHHCHCH2), 4.00 (ddd, 1H, J5–6a = 2.0 Hz, 5 3 J5–6b = 5.3 Hz, J5–4 = 8.8 Hz, H ), 4.00 (dd, 1H, J3–2 = 1.0 Hz, J3–4 = 5.0 Hz, H ), 4.14 (dddd, 1H, J7a–9a = 7a J7a–9b = 1.5 Hz, J7a–8 = 5.0 Hz, J7a–7b = 13.0 Hz, H OCHHCHCH2), 4.21 (dd, 1H, J2–1  J2–3 = 1.0 Hz, 2 4 H ), 4.33 (dd, 1H, J4–3 = 5.0 Hz, J4–5 = 8.8 Hz, H ), 4.43 (d, 1H, J = 11.5 Hz, OCH2C6H5), 4.47 (d, 1H, J = 11.5 Hz, OCH2C6H5), 4.52 (s, 2H, OCH2C6H5), 4.57 (d, 1H, J = 12.2 Hz, OCH2C6H5), 4.67 (d, 1H, J = 1 11.5 Hz, OCH2C6H5), 4.87 (d, 1H, J1–2 = 1.0 Hz, H ), 5.10 (dddd, 1H, J9b–9a = J9b–7a = J9b–7b = 1.5 Hz, 9b J9b–8 = 10.5 Hz, H OCH2CHCHH), 5.22 (dddd, 1H, J9a–9b = J9a–7a = J9a–7b = 1.5 Hz, J9a–8 = 17.0 Hz, 9a 8 H OCH2CHCHH), 5.83 (dddd, 1H, J8–7a = 5.0 Hz, J8–7b = 6.0 Hz, J8–9b = 10.5 Hz, J8–9a = 17.0 Hz, H 13 OCH2CHCH2), 7.24–7.37 (m, 15H, ArH) ppm. C NMR (CDCl3) δ: 68.9, 70.8, 72.0, 72.5, 73.4, 76.7, 77.0, 77.3, 77.3, 78.6, 79.9, 83.0, 107.8, 117.0, 127.6, 127.7, 128.2, 128.3, 128.3, 134.2, 134.3, 138.0, 138.6, 138.9 1 20 ppm. IR (Nujol) ν = 3423, 1462, 1398, 1061 cm− .[α] D = +19.5 (c 0.2, CHCl3). The physico-chemical data are in agreement with those reported in the literature [28]. Pharmaceuticals 2020, 13, 198 9 of 13

Synthesis of 1,2,3,4-tetra-O-acetyl-α-D-xylopyranose (16a). To a solution of 15 (1 g, 6.66 mmol) in pyridine (5 g, 4.5 mL, 62 mmol), Ac2O was added (5.5 g, 4.5 mL, 52.74 mmol). The mixture was stirred at 0 ◦C for 6 h, extracted with Et2O and washed with H2O and CuSO4 saturated solution. The combined organic layers were dried with Na2SO4, filtered and concentrated. Purification of the residue by column chromatography (cyclohexane/EtOAc 9:1) gave 16a as a yellow oil which was dried under N2 + 1 atmosphere because of its thermolability. Yield: 75% (1.59 g, 5 mmol). MS (ESI): 336 (M + NH4 ). H 5b NMR (CDCl3) δ: 2.04–2.19 (m, 12H, CH3CO), 3.72 (dd, 1H, J5b–4 = J5b–5a = 11.0 Hz, H ), 3.95 (dd, 1H, 5a 2 J5a–4 = 6.0 Hz, J5a–5b = 11.0 Hz, H ), 5.04 (dd, 1H, J2–1 = 3.5 Hz, J2–3 = 10.0 Hz, H ), 5.05 (ddd, 1H, J4–5a 4 3 = 6.0 Hz, J4–3 = 10.0 Hz, J4–5b = 11.0 Hz, H ), 5.48 (dd, 1H, J3–2 = J3–4 = 10.0 Hz, H ), 6.27 (d, 1H, J1–2 = 1 13 3.5 Hz, H ) ppm. C NMR (CDCl3) δ: 20.4, 20.6, 20.7, 20.8, 60.6, 68.7, 69.3, 89.2, 168.9, 169.6, 169.7, 1 20 1 13 170.1 ppm. IR (film) ν = 1766, 1232, 1128 cm− .[α] D = +88.0 (c 0.3, CHCl3). H NMR, C NMR, and IR data are in agreement with those reported in the literature [21].

Synthesis of 2,3,4-tri-O-acetyl-D-xylopyranose (17). To a solution of 16a (1 g, 3.14 mmol) in dry DMF (9 mL), AcONH4 (0.485 g, 6.29 mmol) was added and the mixture was stirred at room temperature for 22 h, extracted with EtOAc, and washed with H2O and saturated aq. NH4Cl solution. The combined organic layers were dried with Na2SO4 and concentrated. Purification of the oily residue by column chromatography (cyclohexane/EtOAc 7:3) gave 17 as a white solid. Yield 70% (0.607 g, 2.2 mmol). Mp: + 20 1 142–144 ◦C (EtOAc/petroleum ether). MS (ESI): 299 (M + Na ). [α] D = +69.0 (c 0.2, CHCl3). H NMR, 13C NMR, and IR data are in agreement with those reported in the literature [29].

Synthesis of 2,3,4-tri-O-acetyl-D-xylopyranosyl-α-trichloroacetimidate (14a). To a solution of 17 (0.407 g, 1.48 mmol) and CCl3CN (1.25 g, 0.86 mL, 8.83 mmol) in dry CH2Cl2 (14 mL) at 0 ◦C and under N2 atmosphere, DBU was added (0.09 g, 0.88 mL, 0.59 mmol) and the mixture was stirred at 0 ◦C for 1 h and then concentrated. Purification of the residue by column chromatography (cyclohexane/EtOAc 9:1) gave 14a as a yellow oil (Rf: 0.5, cyclohexane/EtOAc 7:3). Yield: 60% (0.37 g, 0.86 mmol). MS (ESI): + 1 437 (M + NH4 ). H NMR (CDCl3) δ: 2.03 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 5a 5b 3.82 (dd, 1H, J5a–4 = J5a–5b = 11.0 Hz, H ), 4.00 (dd, 1H, J5b–4 = 5.9 Hz, J5b–5a = 11.0 Hz, H ), 5.08 (dd, 2 4 1H, J2–1 = 3.6 Hz, J2–3 = 10.0 Hz, H ), 5.11 (ddd, 1H, J4–5b = 5.9 Hz, J4–5a = 7.5 Hz, J4–3 = 11.0 Hz, H ), 3 1 5.58 (dd, 1H, J3–2 = J3–4 = 10.0 Hz, H ), 6.50 (d, 1H, J1–2 = 3.6 Hz, H ), 8.67 [s, 1H, C(NH)CCl3] ppm. 13 C NMR (CDCl3) δ: 20.5, 20.6, 20.7, 60.8, 68.6, 69.3, 69.9, 76.7, 77.0, 77.3, 93.1, 160.9, 169.8 ppm. IR 1 20 (film): ν = 3481, 2500, 1757, 1487, 1218, 1046 cm− .[α] D = +45.2 (c 0.2, CHCl3). The physico-chemical data are in agreement with those reported in the literature [22].

Synthesis of α-allyl 3,5,6-tri-O-benzyl-(2,3,4-tri-O-acetyl-2-O-β-D-xylopyranosyl)-D-glucofuranoside (19a). A mixture of 12a (0.35 g, 0.71 mmol) and 14a (0.358 g, 0.86 mmol) in dry CH2Cl2 (8.75 mL) under N2 atmosphere and in the presence of activated 4Å molecular sieves (0.8 g) was stirred for 30 min at room temperature, cooled at 20 ◦C and TMSOTf (6 mg, 5 µL, 0.03 mmol) was then added. The − ® mixture was stirred again at room temperature for 6 h, quenched with Et3N, filtered on Celite , and concentrated. Purification of the residue by column chromatography (petroleum ether/Et2O 6:4) gave 19a as a colorless oil (Rf 0.4, petroleum ether/Et2O 1:1). Yield: 45% (0.24 g, 0.32 mmol). MS (ESI): 766 + 1 (M + NH4 ). H NMR (CDCl3) δ: 1.99 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 3.37 5’b 6b (dd, 1H, J5’b–4’ = 7.0 Hz, J5’b–5’a = 12.2 Hz, H ), 3.71 (dd, 1H, J6b–5 = 6.1 Hz, J6b–6a = 10.5 Hz, H ) 3.84 6a 2 5 7b 7a (dd, 1H, J6a–5 = 2.4 Hz, J6a–6b = 10.5 Hz, H ), 3.97–4.05 (m, 3H, H ,H ,H ), 4.18–4.23 (m, 1H, H ), 5’a 3 4.22 (dd, 1H, J5’a–4’ = 4.5 Hz, J5’a–5’b = 12.2 Hz, H ), 4.28 (dd, 1H, J3–2 = 5.0 Hz, J3–4 = 6.5 Hz, H ), 4.33 4 1’ (dd, 1H, J4–3  J4–5 = 6.5 Hz, H ), 4.51–4.58 (m, 5H, OCH2C6H5), 4.63 (d, 1H, J1’–2’ = 5.9 Hz, H ) 4.80 (d, 4’ 1H, J = 11.6 Hz, OCH2C6H5), 4.93 (ddd, 1H, J4’–5’a = 4.5 Hz, J4’–3’  J4’–5’b = 7.0 Hz, H ), 4.99 (dd, 1H, 2’ 1 J2’–1’ = 5.9 Hz, J2’–3’ = 8.0 Hz, H ), 5.02 (d, 1H, J1–2 = 4.3 Hz, H ), 5.12 (dd, 1H, J3’–4’ = 7.0 Hz, J3’–2’ = 3’ 9b 8.0 Hz, H ), 5.16 (dddd, 1H, J9b–9a = J9b–7a = J9b–7b = 1.6 Hz, J9b–8 = 10.5 Hz, H OCH2CHCHH), 5.30 9a (dddd, 1H, J9a–9b = J9a–7a = J9a–7b = 1.6 Hz, J9a–8 = 17.2 Hz, H OCH2CHCHH), 5.89 (dddd, 1H, J8–7a = 8 5.1 Hz, J8–7b = 6.1 Hz, J8–9b = 10.5 Hz, J8–9a = 17.2 Hz, H OCH2CHCH2), 7.26–7.33 (m, 15 H, ArH) Pharmaceuticals 2020, 13, 198 10 of 13

13 ppm. C NMR (CDCl3) δ: 20.6, 20.7, 20.8, 61.7, 68.6, 68.8, 70.1, 70.6, 72.5, 72.6, 73.3, 76.0, 76.7, 76.8, 81.0, 84.9, 99.5, 100.7, 117.0, 122.4, 127.5, 127.6, 128.2, 128.4, 134.2, 137.8, 138.6, 138.8, 169.1, 169.9, 170.0 ppm. 1 20 IR (CHCl3): ν = 3064, 1756, 1454, 1372, 1221, 1057 cm− .[α] D = +17.6 (c = 0.2, MeOH).

Synthesis of β-allyl 3,5,6-tri-O-benzyl-(2,3,4-tri-O-acetyl-2-O-β-D-xylopyranosyl)-D-glucofuranoside (19b). A mixture of 12b (0.35 g, 0.71 mmol) and 14a (0.358 g, 0.86 mmol) in dry CH2Cl2 (8.75 mL) under N2 atmosphere and in the presence of activated 4Å molecular sieves (0.8 g) was stirred for 30 min at room temperature, cooled at 20 ◦C and TMSOTf (6 mg, 5 µL, 0.03 mmol) was then added. The − ® mixture was stirred again at room temperature for 3.5 h, quenched with Et3N, filtered on Celite , and concentrated. Purification of the residue by column chromatography (petroleum ether/Et2O 6:4) gave 19b as a colorless oil (Rf 0.4, petroleum ether/Et2O 1:1). Yield: 60% (0.321 g, 0.43 mmol). MS (ESI): 766 + 1 (M + NH4 ). H NMR (CDCl3) δ: 1.97 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 3.23 5’b 6b (dd, 1H, J5’b–4’ = 9.2 Hz, J5’b–5’a = 12.0 Hz, H ), 3.68 (dd, 1H, J6b–5 = 5.2 Hz, J6b–6a = 10.5 Hz, H ), 3.88 6a 3 (dd, 1H, J6a–5 = 2.0 Hz, J6a–6b = 10.5 Hz, H ), 3.94 (dd, 1H, J3–2 = 1.2 Hz, J3–4 = 5.0 Hz, H ), 3.97 (dddd, 7b 1H, J7b–9a = J7b–9b = 1.5 Hz, J7b–8 = 6.0 Hz, J7b–7a = 13.0 Hz, H , OCHHCHCH2), 4.06 (dd, 1H, J5’a–4’ = 5’a 5 5.2 Hz, J5’a–5’b = 12.0 Hz, H ), 4.06 (ddd, 1H, J5–6a = 2.0 Hz, J5–6b = 5.2, J5–4 = 9.5 Hz, H ), 4.14 (dd, 1H, 2 4 J2–1 = Hz, J2–3 = 1.2 Hz, H ), 4.19 (dd, 1H, J4–3 = 5.0 Hz, J4–5 = 9.5 Hz, H ), 4.19 (dddd, 1H, J7a–9a = 7a 1’ J7a–9b = 1.5 Hz, J7a–8 = 5.0, J7a–7b = 13.0 Hz, H OCHHCHCH2), 4.24 (d, 1H, J1’–2’ = 7.2 Hz, H ), 4.52 (d, 1H, J = 11.2 Hz, OCH2C6H5), 4.53 (d, 1H, J = 12.0 Hz, OCH2C6H5), 4.59 (s, 2H, OCH2C6H5), 4.60 (d, 1H, J = 12.0 Hz, OCH2C6H5), 4.79 (d, 1H, J = 11.2 Hz, OCH2C6H5), 4.83 (dd, 1H, J2’–1’ = 7.2 Hz, J2’–3’ 2’ 4’ = 8.5 Hz, H ), 4.91 (ddd, 1H, J4’–5’a = 5.2 Hz, J4’–3’ = 8.5 Hz, J4’–5’b = 9.2 Hz, H ), 5.01 (d, 1H, J1–2 = 1 3’ 1.2 Hz, H ), 5.08 (dd, 1H, J3’–2’ = J3’–4’ = 8.5 Hz, H ), 5.18 (dddd, 1H, J9b–9a = J9b–9a = J9b–7b = 1.5 Hz, 9b J9b–8 = 10.5 Hz, H OCH2CHCHH), 5.28 (dddd, 1H, J9a–9b = J9a–7a = J9a–7b = 1.5 Hz, J9a–8 = 17.0 Hz, 9a 8 H OCH2CHCHH), 5.89 (dddd, 1H, J8–7a = 5.0 Hz, J8–7b = 6.0 Hz, J8–9b = 10.5 Hz, J8–9a = 17.0 Hz, H 13 OCH2CHCH2), 7.25–7.35 (m, 15 H, ArH) ppm. C NMR (MeOD) δ: 19.1, 19.1, 19.3, 61.9, 68.5, 68.8, 69.8, 70.9, 71.8, 71.8, 71.8, 73.0, 76.4, 79.9, 80.4, 84.6, 100.1, 106.6, 115.7, 117.1, 127.2, 127.4, 127.4, 127.5, 127.8, 127.9, 127.9, 128.1, 134.2, 137.9, 138.4, 138.7, 169.6, 170.0, 170.2 ppm. IR (CHCl3): ν = 3064, 1756, 1454, 1372, 1221, 1057 cm 1.[α]20 = 12.8 (c 0.2, MeOH). − D − Synthesis of β-allyl 3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-D-glucofuranoside (22b). To a solution of 19b (0.21 g, 0.29 mmol) in dry MeOH (62 mL) MeONa was added (0.038 g, 0.70 mmol) and the mixture was stirred at room temperature for 3 h, then concentrated. Purification of the residue by column chromatography (cyclohexane/EtOAc 1:9) gave 22b as a pale-yellow oil (Rf: 0.3, EtOAc). Yield: 90% + 1 (0.161 g, 0.26 mmol). MS (ESI): 605 (M + H ). H NMR (CDCl3) δ: 2.51 (br s, 1H, OH), 2.77 (br s, 1H, 5’b OH), 2.94 (br s, 1H, OH), 3.19 (dd, 1H, J5’b-4’ = 9.2, J5’b-5’a = 12.0 Hz, H ), 3.29 (dd, 1H, J2’-1’ = 6.5 Hz, 2’ 3’ J2’-3’ = 8.0 Hz, H ), 3.43 (dd, 1H, J3’-2’ = J3’-4’ = 8.0 Hz, H ), 3.67 (ddd, J4’-5’a = 4.8 Hz, J4’-3’ = 8.0 Hz, 4’ 6b J4’-5’b = 9.2 Hz, H ), 3.70 (dd, 1H, J6b-5 = 4.5 Hz, J6b–6a = 10.8 Hz, H ), 3.88 (dd, 1H, J6a–5 = 2.0 Hz, 6a 5’a J6a–6b = 10.8 Hz, H ), 3.95 (dd, 1H, J5’a–4’ = 4.8 Hz, J5’a–5’b = 12.0 Hz, H ), 3.98 (dddd, 1H, J7b–9a = 7b J7b–9b = 1.5 Hz, J7b–8 = 6.0 Hz, J7b–7a = 13.0 Hz, H OCHHCHCH2), 4.04 (dd, 1H, J3–2 = 1.0 Hz, J3–4 3 5 = 4.8 Hz, H ), 4.06 (ddd, 1H, J5–6a = 2.0 Hz, J5–6b = 4.5 Hz, J5–4 = 9.2 Hz, H ), 4.08 (d, 1H, J1’–2’ = 6.5 1’ 7a Hz, H ), 4.19 (dddd, 1H, J7a–9a = J7a–9b = 1.5 Hz, J7a–8 = 5.0 Hz, J7a–7b = 13.0 Hz, H OCHHCHCH2), 2 4 4.23 (dd, 1H, J2–1 = J2–3 = 1.0 Hz, H ), 4.35 (dd, 1H, J4–3 = 4.8 Hz, J4–5 = 9.2 Hz, H ), 4.51 (d, 1H, J = 11.2 Hz, OCH2C6H5), 4.56 (d, 1H, J = 12.5 Hz, OCH2C6H5), 4.58 (d, 1H, J = 12.5 Hz, OCH2C6H5), 4.61 (d, 1H, J = 12.5 Hz, OCH2C6H5), 4.62 (d, 1H, J = 12.5 Hz, OCH2C6H5), 4.75 (d, 1H, J = 11.2 Hz, 1 OCH2C6H5), 5.06 (d, 1H, J1-2 = 1.0 Hz, H ), 5.17 (dddd, 1H, J9b–9a = J9b–7a = J9b–7b = 1.6 Hz, J9b–8 = 9b 9a 10.5 Hz, H OCH2CHCHH), 5.28 (dddd, 1H, J9a–9b = J9a–7a = J9a–7b = 1.6 Hz, J9a–8 = 17.0 Hz, H 8 OCH2CHCHH), 5.90 (dddd, 1H, J8–7a = 5.0 Hz, J8–7b = 6.0 Hz, J8–9b = 10.5 Hz, J8–9a = 17.0 Hz, H 13 OCH2CHCH2), 7.28–7.37 (m, 15H, ArH) ppm. C NMR (CDCl3) δ: 64.7, 68.9, 69.4, 70.3, 72.4, 72.4, 72.4, 72.6, 73.4, 75.1, 79.9, 80.5, 84.6, 101.9, 106.6, 117.0, 127.5, 127.6, 127.7, 127.7, 127.9, 128.0, 128.3, 128.3, Pharmaceuticals 2020, 13, 198 11 of 13

1 20 128.4, 134.1, 137.7, 138.7, 138.7 ppm. IR (CHCl3): ν = 3415, 3019, 1785, 1450, 1368, 1045 cm− .[α] D = –19.6 (c = 0.2, MeOH).

Synthesis of 3,5,6-tri-O-benzyl-2-O-β-D-xylopyranosyl-α-D-glucofuranoside and 3,5,6-tri-O-benzyl-2-O -β-D-xylopyranosyl-β-D-glucofuranoside (23). To a solution of 22b (0.07 g, 0.12 mmol) in MeCOONa (0.034 g, 0.42 mmol) and MeCOOH (0.22 mL), PdCl2 (0.04 g, 0.22 mmol), and H2O (11 µL) were added. The mixture was stirred at room temperature for 12 h, filtered on Celite®, and washed with EtOAc. The filtrate was washed with a saturated NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by column chromatography (EtOAc/cyclohexane 8:2) gave 23 (α/β 2:3) as a colorless oil (Rf: 23a 0.2, 23b + 1 0.3; EtOAc/MeOH 99:1). Yield: 42% (0.027 g, 0.05 mmol). MS (ESI): 600 (M + NH4 ). H NMR (MeOD) δ: 3.11–3.21 (m, 3H), 3.23–3.30 (m, 3H), 3.44–3.54 (m, 3H), 3.66–3.73 (m, 2H), 3.82–3.91 (m, 3H), 3.93–3.98 (m, 2H), 4.08–4.11 (m, 1H), 4.11–4.14 (4H), 4.19 (dd, 1H, J1 = 2.6 Hz, J2 = 4.0 Hz), 4.25 (dd, 1H, J1 = 2.5 Hz, J2 = 4.6 Hz), 4.28 (d, 1H, J = 4.0 Hz), 4.30 (dd, 1H, J1 = J2 = 2.1 Hz), 4.38 (dd, 1H, J1 = 4.6 Hz, J2 = 1 7.9 Hz), 4.50–4.77 (m, 10H, OCH2C6H5), 5.25 (d, 1H, J = 1.0 Hz, H β-anomer), 5.39 (d, 1H, J = 4.0 Hz, H1 α-anomer), 7.26–7.38 (m, 30H, ArH) ppm. 13C NMR (MeOD) δ: 65.6, 65.6, 69.7, 69.7, 70.2, 70.2, 71.8, 71.9, 71.9, 71.9, 72.9, 73.0, 73.0, 73.3, 75.9, 76.4, 76.5, 76.9, 79.8, 80.7, 80.8, 81.6, 85.0, 85.0, 96.5, 102.3, 102.8, 103.0, 127.1, 127.1, 127.2, 127.3, 127.3, 127.4, 127.4, 127.4, 127.5, 127.7, 127.8, 127.8, 127.9, 127.9, 128.0, 128.0, 128.1, 128.1, 137.9, 138.0, 138.4, 138.4, 138.8, 138.8 ppm. IR (Nujol): ν = 3399, 1458, 1376, 1041 cm 1.[α]20 = 32.6 (c = 0.3, MeOH). − D − Synthesis of 2-O-β-D-xylopyranosyl-D-α-glucopyranose and 2-O-β-D-xylopyranosyl-D-β-glucopyranose (8, sambubiose). To a solution of 23 (0.09 g, 0.15 mmol) in EtOH (9 mL), Pd/C 10% (0.018 g) was added and the mixture was hydrogenated under stirring at room temperature at 1 atm for 6 h, then filtered on Celite®, washed with EtOH, and kept overnight. The crystals obtained were then filtered and triturated to obtain 8 (sambubiose) as a white solid (Rf: 0.2, CH3CN/H2O 9:1). Yield 51% (0.024 g, 0.08 1 6 mmol). Mp: 132–134 ◦C (petroleum ether). MS (ESI): 313 (M + 1), 311 (M-1). H NMR (DMSO-d ) δ: 3.02–3.05 (m, 2H, H5,H2’), 3.08–3.17 (m, 3H, H2,H3’,H5’a), 3.23–3.28 (m, 1H, H4’), 3.41–3.47 (m, 1H, 6a 3 4 6b 5’b 1’ H ), 3.54–3.68 (m, 4H, H ,H ,H ,H ), 4.25 (d, 1H, J1’–2’ = 7.2 Hz, H ), 4.39 (dd, 1H, JOH6–6a  6 JOH6–6b = 5.5 Hz, OH ), 4.77 (d, 1H, J = 3.2 Hz, OH), 4.93 (d, 1H, J = 5.6 Hz, OH), 4.98 (d, 1H, J = 5.2 Hz, OH4’), 5.00 (d, 1 H, J = 4.8 Hz, OH), 5.02–5.05 (m, 2H, H1, OH) 6.34 (d, 1H, J = 4.5 Hz, OH1) ppm. 13C NMR (DMSO-d6) δ: 61.5 (C6), 66.1 (C5’), 69.9 (C4’), 70.5 (C5), 72.1 (C3), 72.4 (C4), 74.2 (C2’), 76.7 (C3’), 13 82.7 (C2), 92.0 (C1), 106.4 (C1’). α-anomer C NMR (D2O) δ: 60.4 (C6), 65.0 (C5’), 69.2 (C4’), 69.4 (C5), 13 71.0 (C3), 71.7 (C4), 73.1 (C2’), 75.5 (C3’), 80.7 (C2), 91.7 (C1), 104.7 (C1’). β-anomer C NMR (D2O) δ: 60.6 (C6), 65.1 (C5’), 69.1 (C4’), 69.4 (C5), 73.4 (C2’), 75.5, 75.69, 75.64, 81.8 (C2), 94.6 (C1), 103.6 (C1’). IR (Nujol): ν = 3400, 2840, 1070 cm 1.[α]20 = 5.0 (c = 0.03, MeOH). [10] − D − 4. Conclusions An efficient method for the total synthesis of natural disaccharide sambubiose was developed. Our investigation enabled us to identify the best combination of donor and acceptor glycosides as well as a catalyst that allowed a complete stereocontrol of the key O-glycosylation step. The protocol described herein was carried out using stable, safe, non-toxic agents and a step-by-step economical synthetic route. At the same time, a satisfactory overall yield and the characterization of sambubiose were achieved, thus overcoming the drawbacks associated with the current method reported in the literature [10].

Author Contributions: Conceptualization, S.L. and A.D.; methodology, S.L. and A.D.; validation, S.L. and A.D.; formal analysis, S.L. and A.D.; investigation, M.G.C. and P.M.; resources, S.L. and A.D.; data curation, S.L. and A.D.; writing—original draft preparation, S.L. and A.D.; writing—review and editing, M.G.C. and P.M.; visualization, S.L. and A.D.; supervision, S.L. and A.D.; project administration, S.L. and A.D.; funding acquisition, S.L. and A.D. All authors have read and agreed to the published version of the manuscript. Pharmaceuticals 2020, 13, 198 12 of 13

Funding: This research was funded by the University of Urbino Carlo Bo. Acknowledgments: The authors wish to thank Giovanni Piersanti for his helpful suggestions. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Landis-Piwowar, K.R.; Iyer, N.R. Cancer chemoprevention: Current state of the art. Cancer Growth Metastasis 2014, 7, 19–25. [CrossRef][PubMed] 2. Gentili, B.; Horowitz, R.M. Flavonoids of citrus. IX. Some new C-glycosylflavones and nuclear magnetic resonance method for differentiating 6- and 8-C-glycosyl isomers. J. Org. Chem. 1968, 33, 1571–1577. [CrossRef] 3. Gil, M.; Ferreres, F.; Tomás-Barberán, F.A. Effect of modified atmosphere packaging on the flavonoids and vitamin C content of minimally processed Swiss chard (Beta vulgaris subspecies cycla). J. Agric. Food Chem. 1998, 46, 2007–2012. [CrossRef] 4. Sato, F.; Matsukawa, Y.; Matsumoto, K.; Nishino, H.; Sakai, T. Apigenin induces morphological differentiation and G2-M arrest in rat neuronal cells. Biochem. Biophys. Res. Commun. 1994, 204, 578–584. [CrossRef] 5. Reichel, L.; Reichwald, W. Ueber die farbstoffe der schawarzen holunderbeere. Naturwissenschaften 1960, 47, 40–41. [CrossRef] 6. Olejnik, A.; Kowalska, K.; Olkowicz, M.; Rychlik, J.; Juzwa, W.; Myszka, K.; Dembczynski, R.; Bialas, W. Anti-inflammatory effects of gastrointestinal digested Sambucus nigra L. fruit extract analysed in co-cultured intestinal epithelial cells and lipopolysaccharide-stimulated macrophages. J. Funct. Foods 2015, 19, 649–660. [CrossRef] 7. Loizzo, M.R.; Pugliese, A.; Bonesi, M.; Tenuta, M.C.; Menichini, F.; Xiao, J.; Tundis, R. Edible flowers: A rich source of phytochemicals with antioxidant and hypoglycemic properties. J. Agric. Food Chem. 2016, 64, 2467–2474. [CrossRef] 8. Wu, X.; Pittman, H.E., III; Prior, R.L. Fate of anthocyanins and antioxidant capacity in contents of the gastrointestinal tract of weanling pigs following black raspberry consumption. J. Agric. Food Chem. 2006, 54, 583–589. [CrossRef] 9. Socaciu, C. Food Colorants. Chemical and Functional Properties, 1st ed.; CRC Press: Boca Raton, FL, USA, 2007; p. 648. 10. Erbing, B.; Lindberg, B. Synthesis of sambubiose. Acta Chem. Scand. 1969, 23, 2213–2215. [CrossRef] 11. Dick, W.R., Jr.; Hodge, J.E.; Inglett, G.E. Structure-taste relationships in (1 2)-linked disaccharides. Carbohydr. → Res. 1974, 36, 319–329. [CrossRef] 12. Helferich, B.; Ost, W. Synthese einiger β-d-xylopyranoside. Chem. Ber. 1962, 95, 2612–2615. [CrossRef] 13. Lucarini, S.; Fagioli, L.; Campana, R.; Cole, H.; Duranti, A.; Baffone, W.; Vllasaliu, D.; Casettari, L. Unsaturated fatty acids lactose esters: Cytotoxicity, permeability enhancement and antimicrobial activity. Eur. J. Pharm. Biopharm. 2016, 107, 88–96. [CrossRef][PubMed] 14. Perinelli, D.R.; Lucarini, S.; Fagioli, L.; Campana, R.; Vllasaliu, D.; Duranti, A.; Casettari, L. Lactose oleate as new biocompatible surfactant for pharmaceutical applications. Eur. J. Pharm. Biopharm. 2018, 124, 55–62. [CrossRef][PubMed] 15. Lucarini, S.; Fagioli, L.; Cavanagh, R.; Liang, W.; Perinelli, D.; Campana, M.; Stolnik, S.; Lam, J.; Casettari, L.; Duranti, A. Synthesis, structure–activity relationships and in vitro toxicity profile of lactose-based fatty acid monoesters as possible drug permeability enhancers. Pharmaceutics 2018, 10, 81. [CrossRef] 16. Campana, R.; Merli, A.; Verboni, M.; Biondo, F.; Favi, G.; Duranti, A.; Lucarini, S. Synthesis and evaluation of saccharide-based aliphatic and aromatic esters as antimicrobial and antibiofilm agents. Pharmaceuticals 2019, 12, 186. [CrossRef] 17. Das, S.N.; Chowdhury, A.; Tripathi, N.; Jana, P.K.; Mandal, S.B. Exploitation of in situ generated sugar-based olefin keto-nitrones: Synthesis of carbocycles, heterocycles, and nucleoside derivatives. J. Org. Chem. 2015, 80, 1136–1148. [CrossRef] 18. Mukaiyama, T.; Murai, Y.; Shoda, S. An efficient method for glucosylation of hydroxy compounds using glucopyranosyl fluoride. Chem. Lett. 1981, 10, 431–432. [CrossRef] 19. Schmidt, R.R.; Kinzy, W. Anomeric-oxygen activation for glycoside synthesis: The trichloroacetimidate method. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21. Pharmaceuticals 2020, 13, 198 13 of 13

20. Hayashi, M.; Hashimoto, S.-I.; Noyori, R. Simple synthesis of glycosyl fluorides. Chem. Lett. 1984, 13, 1747–1750. [CrossRef] 21. Kam, B.L.; Barascut, J.-L.; Imbach, J.-L. A general method of synthesis and isolation, and an NMR-spectroscopic study, of tetra-O-acetyl-d-aldopentofuranoses. Carbohydr. Res. 1979, 69, 135–142. [CrossRef] 22. Mori, M.; Ito, Y.; Ogawa, T. Total synthesis of the mollu-series glycosyl ceramides α-d-Manp-(1 3)-β-d-Manp-(1 4)-β-d-Glcp-(1 1)-Cer and α-d-Manp-(1 3)-[β-d-Xylp-(1 2)]-β-d- → → → → → Manp-(1 4)-β-d-Glcp-(1 1)-Cer. Carbohydr. Res. 1990, 195, 199–224. [CrossRef] → → 23. Chang, C.-W.T.; Hui, Y.; Elchert, B.; Wang, J.; Li, J.; Ra, R. Pyranmycins, a novel class of aminoglycosides with improved acid stability: The SAR of d-pyranoses on ring III of pyranmycin. Org. Lett. 2002, 26, 4603–4606. [CrossRef][PubMed] 24. Kumar, A.; Doddi, V.R.; Vankar, Y.D. Mild and efficient chemoselective deprotection of anomeric O-methyl glycosides with trityl tetrafluoroborate. J. Org. Chem. 2008, 73, 5993–5995. [CrossRef][PubMed] 25. Hohgardt, H.; Dietrich, W.; Kühne, H.; Müller, D.; Grzelak, D.; Welzel, P.Synthesis of two structural analogues of the smallest antibiotically active degradation product of moenomycin a. Tetrahedron 2008, 44, 5771–5790. [CrossRef] 26. Maranduba, A.; Veyrieres, A. Glycosylation of lactose. Synthesis of methyl O-(2-acetamido-2-deoxy-β-d- glucopyranosyl)-(1 3)-O-β-d-galactopyranosyl-(1 4)-β-d-glucopyranoside and methyl O-β-d-galactop → → yranosyl-(1 4)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1 3)-O-β-d-galactopyranosyl-(1 4)-β-d- → → → glucopyranoside. Carbohydr. Res. 1985, 135, 330–336. 27. Boonyarattanakalin, S.; Michieletti, M.; Lepenies, B.; Seeberger, P.H. Chemical synthesis of all phosphatidylinositol mannoside (PIM) glycans from mycobacterium tuberculosis. J. Am. Chem. Soc. 2008, 130, 16791–16799. [CrossRef] 28. Listkowski, A.; Ing, P.; Cheaib, R.; Chambert, S.; Doutheau, A.; Queneau, Y. Carboxymethylglycoside lactones (CMGLs): Structural variations on the carbohydrate moiety. Tetrahedron: Asymmetry 2007, 18, 2201–2210. [CrossRef]

29. Yan, Y.-L.; Guo, J.-R.; Liang, C.-F. Sequential Dy(OTf)3-catalyzed solvent-free per-O-acetylation and regioselective anomeric de-O-acetylation of carbohydrates. Chem. Asian J. 2017, 12, 2471–2479. [CrossRef]

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