An Optimized Facile Procedure to Synthesize and Purify Allicin

molecules

Article An Optimized Facile Procedure to Synthesize and Purify Allicin

Frank Albrecht 1,†, Roman Leontiev 1,2,†, Claus Jacob 2 and Alan J. Slusarenko 1,*

1 Department of Plant Physiology, RWTH Aachen University, D-52056 Aachen, Germany; [email protected] (F.A.); [email protected] (R.L.) 2 Division of Bioorganic Chemistry, School of Pharmacy, Campus B2 1, University of Saarland, D-66123 Saarbruecken, Saarland, Germany; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work.

Academic Editors: Thomas J. Schmidt and Derek J. McPhee Received: 1 March 2017; Accepted: 5 May 2017; Published: 10 May 2017

Abstract: Allicin is a reactive sulfur species (RSS) and defence substance from garlic (Allium sativum L.). The compound is a broad-spectrum antibiotic that is also effective against multiple drug resistant (MDR) strains. A detailed protocol for allicin synthesis based on diallyl-disulfide (DADS) oxidation by H2O2 using acetic acid as a catalyst was published in 2001 by Lawson and Wang. Here we report on improvements to this basic method, clarify the mechanism of the reaction and show that it is zero-order with respect to DADS and first-order with respect to the concentration of H2O2. The progress of allicin synthesis and the reaction mechanism were analyzsd by high-performance liquid chromatography (HPLC) and the identity and purity of the products was verified with LC-MS and 1H-NMR. We were able to obtain allicin of high purity (>98%) and >91% yield, with standard equipment available in any reasonable biological laboratory. This protocol will enable researchers to prepare and work with easily and cheaply prepared allicin of high quality.

Keywords: allicin; Allium sativum; diallyl-disulfide; catalytic oxidation; reactive sulfur species; dipropyl-disulfide; thiosulfinate

1. Introduction The sulfur-containing compound allicin (2-Propene-1-sulfinothioic acid S-2-propenyl ester, or diallyl-thiosulfinate, DATS) is produced in damaged tissue of garlic (Allium sativum), ramsons (Allium ursinum), and hooker chives (Allium hookeri) and gives these plants their typical odours [1]. Garlic is highly valued in the cuisines of many nations because of its excellent flavour and its pungent smell. Additionally, it has long been believed that allicin, or at least garlic consumption, is beneficial to health [2]. In 1944 Cavallito and Bailey demonstrated that allicin inhibited the growth of Staphylococcus aureus and other bacteria in liquid culture [3]. Furthermore, allicin was shown to induce apoptosis, often selectively, in mammalian cancer cells cultured in vitro [4,5], in intact tissues in vivo [6], and in cells of yeast (Saccharomyces cerevisiae), a model fungal eucaryote [7]. These properties turn allicin into a highly interesting compound for clinical investigations. Stoll and Seebeck first reported the synthesis of allicin in 1947, but without specifying experimental details [8]. Their chemical synthesis of allicin was based on the oxidation of diallyl-disulfide (DADS) by peracetic acid as a mild oxidizing agent. A more detailed protocol of this basic method was published by Lawson and Koch in 1994 and Lawson and Wang in 2001 [9,10]. Other methods to synthesize allicin utilizing magnesium monoperoxyphthalate [11] or chloroperbenzoic acid have also been reported [12,13]. Nevertheless, it is still challenging to obtain pure allicin in acceptable yields.

Molecules 2017, 22, 770; doi:10.3390/molecules22050770 www.mdpi.com/journal/molecules Molecules 2017, 22, 770 2 of 13

In the original protocol, DADS was stirred into a mixture of acetic acid and H O and incubated Molecules 2017, 22, 770 2 2 2 of 14 at room temperature (RT) for 4 h with constant stirring. The reaction was stopped by adding

five volumesIn the oforiginal water protocol, and extracted DADS was with stirre dichloromethaned into a mixture of (DCM) acetic acid to retrieveand H2O allicin2 and incubated along with unreactedat room DADS, temperature some acetic (RT) acid,for 4 andh with DCM-soluble constant stirring. reaction The byproducts. reaction was The stopped lipophilic by adding undissociated five acidvolumes catalyst of in water the DCMand extracted phase with was dichloromethan neutralized withe (DCM) aqueous to retrieve sodium allicin carbonate along with solution unreacted which facilitatedDADS, partitioning some acetic ofacid, the and hydrophilic DCM-soluble sodium reaction acetate byproducts. generated The into lipophilic the aqueous undissociated phase. DCM acid was removedcatalyst by in rotary the DCM evaporation phase was at neutralized RT at reduced with aq pressureueous sodium to yield carbonate an oily residuesolution ofwhich allicin, facilitated unreacted DADS,partitioning and byproducts. of the hydrophilic Further sodium purification acetate generated of allicin into was the based aqueous on thephase. differential DCM was partitioningremoved by rotary evaporation at RT at reduced pressure to yield an oily residue of allicin, unreacted DADS, of the constituents of the oily residue between n-hexane and an aqueous phase (two washes). and byproducts. Further purification of allicin was based on the differential partitioning of the Unreacted DADS and some allicin accumulated in the n-hexane phase, but allicin, which is more polar constituents of the oily residue between n-hexane and an aqueous phase (two washes). Unreacted thanDADS DADS, and concentrated some allicin to accumulated some extent in in the the n aqueous-hexane phase, phase. but The allicin, separation which method is more waspolar inefficient, than however,DADS, and concentrated allicin losses to some occurred extent at in this the stage.aqueou Finally,s phase. the The allicin-containing separation method aqueous was inefficient, phasewas partitionedhowever, against and allicin DCM losses to isolate occurred allicin at this and stage. dried Finally, over anhydrousthe allicin-containing CaSO4. Allicin aqueous was phase obtained was as an oilypartitioned residue against after evaporation DCM to isolate of the allicin DCM and under dried reducedover anhydrous pressure CaSO at RT.4. Allicin was obtained as anThis oily synthesis residue after consists evaporation of at least of the two DCM reaction under reduced steps. Firstly, pressure the at organicRT. peracid is formed by oxidationThis of thesynthesis organic consists acid by of Hat2 Oleast2. Secondly, two reaction DADS steps. is oxidizedFirstly, the by organic the peracid, peracid thus is formed regenerating by the parentoxidation organic of the organic acid. It acid has by been H2O reported2. Secondly, that DADS peracids, is oxidized such by as the performic peracid, thus and regenerating peracetic acids, the parent organic acid. It has been reported that peracids, such as performic and peracetic acids, are are adequately soluble in the organic phase [14], but DADS is immiscible with the aqueous H2O2 solutionadequately and the soluble reactions in the therefore organic phase take place[14], but in DADS a two phaseis immiscible system. with the aqueous H2O2 solution and the reactions therefore take place in a two phase system. In the optimized method described in this paper we used a formic acid catalyst instead of acetic In the optimized method described in this paper we used a formic acid catalyst instead of acetic acid, which enabled us to carry out the reaction at 0 ◦C under more controlled conditions and we acid, which enabled us to carry out the reaction at 0 °C under more controlled conditions and we systematicallysystematically varied varied the the concentrations concentrations of of the the reactants,reactants, while while following following the the progress progress of the of thereaction reaction usingusing HPLC. HPLC. Furthermore, Furthermore, we we developed developed aa silicasilica gel column chromatography chromatography protocol protocol for forallicin allicin purificationpurification which which avoided avoided the the losses losses associated associated withwith the original solvent solvent partitioning partitioning procedure. procedure. A reactionA reaction mechanism mechanism for for Stoll Stoll and and Seebeck’sSeebeck’s synt synthesishesis was was postulated postulated by byNikolic Nikolic et al. et [15] al. [15] proposingproposing oxidative oxidative cleavage cleavage of theof the S–S S–S bond bond in DADS in DADS by hydroxyl-radicalsby hydroxyl-radicals generated generated from from the the acidic H2Oacidic2 to give H2O allyl-sulfenic2 to give allyl-sulfenic acid which acid condenses which condenses to yield to allicin yield (Schemeallicin (Scheme1). In contrast, 1). In contrast, an alternate an mechanism,alternate namelymechanism, direct namely oxidation direct ofoxidation one of theof one S-atoms of the in S-atoms DADS withoutin DADS oxidativewithout oxidative cleavage of the S–Scleavage bond, of isthe also S–S plausible bond, is also (Scheme plausible1). (Scheme Here we 1). provide Here we data provide supporting data supporting an oxidative an oxidative cleavage mechanismcleavage andmechanism condensation and condensation of two sulfenic of two sulfenic acid molecules acid molecules to yield to yield allicin, allicin, but but without without a needa need for hydroxyl-radicals. for hydroxyl-radicals.

SchemeScheme 1. Allicin 1. Allicin synthesis synthesis from from diallyl-disulﬁde diallyl-disulfide (DADS) through through oxidation oxidation by by a peracid a peracid generated generated

withwith H2O H22inO2 thein the reaction reaction mixture. mixture. The The organic organic acidacid serves serves as as an an intermediate intermediate catalyst. catalyst.

2. Results2. Results and and Discussion Discussion

2.1. Comparison2.1. Comparison of DADSof DADS Oxidation Oxidation Catalyzed Catalyzedby by AceticAcetic Acid or or Formic Formic Acid Acid PreliminaryPreliminary experiments experiments substituting substituting formicformic acid for for acetic acetic acid acid at at RT RT resulted resulted in inrapid rapid overheating overheating of theof reactionthe reaction mixture mixture accompanied accompanied by by massive massive byproductbyproduct formation, formation, therefore, therefore, we we carried carried out outthe the

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Moleculesformic acid2017, catalyzed22, 770 allicin syntheses at 0 °C. The progress of the oxidation reactions was followed3 of 13 by HPLC analysis; i.e., disappearance of the DADS peak and appearance of the allicin peak. In a first formicattempt, acid we catalyzed withdrew allicin small syntheses samples of at 0the◦C. procee The progressding reaction, of the oxidationdiluted them reactions with methanol was followed and bymeasured HPLC analysis;the amount i.e., of disappearance allicin and DADS. of the This DADS approach peak andwas appearancenot reliable, ofhowever, the allicin due peak. to the Infact a firstthat attempt,the reaction we withdrewmixture was small an emulsion. samples of It the was, proceeding therefore,reaction, difficult, diluteddespite themthorough with mixing, methanol to andguarantee measured the same the amount distribution of allicin of content and DADS. in withdrawn This approach samples was and not the reliable, remainder however, of the due reaction to the factmix thatin the the flask. reaction Measurements mixture was confirmed an emulsion. these It co was,ncerns therefore, and showed difficult, unrealistic despite thoroughkinetics (data mixing, not toshown). guarantee Therefore, the same in distributiona second approach, of content the in reaction withdrawn was samplescarried out and in the several remainder parallel of thealiquots reaction on mixa micro-scale in the flask. and Measurements each aliquot was confirmed diluted with these methan concernsol as and a whole showed to give unrealistic a single kinetics data point. (data Thus, not shown).every time Therefore, point presented in a second in Fig. approach, 1 shows the an reaction independent was carried parallel out reaction in several run. parallel Reaction aliquots progress on awas micro-scale followed and by eachcalculating aliquot the was percentage diluted with ratio methanol of actual as a allicin whole yield to give divided a single by data the point. theoretical Thus, everymaximum time pointyield presented(100% of DA in Fig.DS 1converted shows an to independent allicin) to indicate parallel reactionthe percent run. conversion Reaction progress during was the followedcourse of by the calculating reaction. Despite the percentage the lower ratio reaction of actual temperature, allicin yield allicin divided was by formed the theoretical more rapidly maximum and yieldto a greater (100% ofyield DADS (78% converted conversion to allicin) by 4 h) to with indicate formic the percentacid as conversioncatalyst than during with theacetic course acid of (58% the reaction.conversion Despite at 4 h) the(Figure lower 1). reaction temperature, allicin was formed more rapidly and to a greater yield (78% conversion by 4 h) with formic acid as catalyst than with acetic acid (58% conversion at 4 h) (Figure1).

100 90 80 70 60 50 40

conversion (%) 30 Acetic acid 20 Formic acid 10 0 012345678 time (h)

Figure 1. Kinetics of allicin synthesis. The oxidation of DADS to allicin was catalyzed by either aceticFigure acid 1. Kinetics at 20 ◦C of or allicin formic synthesis. acid at 0The◦C. oxidation All reactions of DADS took placeto allicin in sealedwas catalyzed 2 mL reaction by either tubes acetic in temperature-controlledacid at 20 °C or formic rotary acid shakers at 0 °C. and All via reactions continual took shaking place at 1200in sealed rpm to2 ensuremL reaction optimal tubes mixing. in Thetemperature-controlled products were separated rotary by HPLCshakers and and quantiﬁed via continual with a shaking UV detector at 1200 at 254 rpm nm. to Reaction ensure progressoptimal wasmixing. followed The products by calculating were separated the percentage by HPLC ratio and ofqu actualantified allicin with a yield UV detector divided at by 254 the nm. theoretical Reaction maximumprogress was yield followed to indicate by thecalculating percentage the of percentage conversion ratio during of theactual course allicin of the yield reaction. divided by the theoretical maximum yield to indicate the percentage of conversion during the course of the reaction. Byproducts detectable by HPLC and presumably arising via decomposition, were observed increasinglyByproducts with detectable incubation by times HPLC longer and presumably than 4 h. Quantitativelyarising via decomposition, slightly lower were amounts observed of byproductsincreasingly werewith observedincubation at 0times◦C withlonger formic than acid 4 h. as Quantitatively a catalyst than slightly with acetic lower acid amounts at 20 ◦ofC (Figurebyproducts2). were observed at 0 °C with formic acid as a catalyst than with acetic acid at 20 °C (Figure 2). LesserLesser byproduct formation using using formic formic acid acid at at 0 0°C◦C as as shown shown in inFigure Figure 2 2may may be be explained explained by byallicin’s allicin’s increased increased reactivity reactivity and andinherent inherent instabilit instabilityy at higher at higher temperatures. temperatures. The instability The instability of allicin of allicinat higher at highertemperatures temperatures was reported was reported to be increased to be increased by hydrophobic by hydrophobic solvents solventssuch as any such residual as any residualDADS [16]. DADS For [ 16those]. For reasons, those reasons, the reaction the reaction should should be stopped be stopped at the atlatest the latestafter after4 h, by 4 h, adding by adding five volumes of H2O, even though conversion is incomplete. Furthermore, if not tempered to 20 ◦°C ﬁve volumes of H2O, even though conversion is incomplete. Furthermore, if not tempered to 20 C differentdifferent RTsRTs will will lead lead to differentto different kinetics kinetics for the for reaction the reaction and the and need the for need new calibrations.for new calibrations. Therefore,

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Therefore, we propose that it is advantageous for reasons of increased yield and reaction consistency we propose that it is advantageous for reasons of increased yield and reaction consistency to use formic toto useuse formicformic acidacid asas aa catalystcatalyst andand toto carrycarry outout thethe reactionreaction onon iceice atat 00 °C.°C. acid as a catalyst and to carry out the reaction on ice at 0 ◦C.

1200 Acetic acid 1000 Formic acid (254 nm) (254 nm) 800

600

400

200 relative absorbance relative absorbance 0 0 2 4 6 8 10121416182022242628303234 timetime (min)(min)

FigureFigure 2.2. ComparisonComparison of representativerepresentative end point tracestraces ofof thethe productsproducts ofof routineroutineroutine allicinallicinallicin synthesessynthesessyntheses withwith eithereither aceticacetic oror formicformic acids.acids.

2.2.2.2. Reaction Order with Respect toto IndividualIndividual ReactantsReactants TheThe kinetics shown in in Figure Figure 11 notnot onlyonly reveal a faster faster reaction reaction whenwhen when formicformic formic acidacid acid isis is usedused used asas as aa acatalyst, catalyst, but but also also give give information information about about the the reacti reactionon order. order. Thus, Thus, after after 2 2 h—48%, h—48%, after 4 h—76% and afterafter 66 h—90%h—90% ofof thethe DADS DADS was was converted converted to to allicin. allicin. This This is is an an approximate approximate halving halving of of the the amount amount of DADSof DADS every every 2 h 2 indicating h indicating that that the the overall overall reaction reactiontion followed followedfollowed ﬁrst firstfirst order orderorder kinetics. kinetics.kinetics. We WeWe investigated investigatedinvestigated the reactionthethe reactionreaction kinetics kineticskinetics in more inin moremore detail detaildetail and andand showed showedshowed that ththat the the shaking shaking conditions conditions for for the the two two phasephase reactionreaction werewere aa limitinglimiting factorfactor forfor thethe reactionreaction speedspeed (Figure(Figure3 ).3). TheThe reactionreaction raterate cancan bebe seenseen toto increase increase proportionallyproportionally upup toto 12001200 rpm,rpm, whichwhich waswas thusthus chosen as the routine routine shaking shaking velocity velocity for for micro-scale micro-scale synthesissynthesis reactions.reactions.

30 25 20 15 10 conversion (%) conversion (%) 5 0 0 500 1000 1500 shaking velocity (rpm)

FigureFigure 3.3. The TheThe effect effecteffect of of shakingof shakingshaking velocity velocityvelocity on the onon reaction thethe reacreac rate.tiontion Micro-scale rate.rate. Micro-scaleMicro-scale reactions reactionsreactions using a pre-incubated usingusing aa pre-pre- ◦ 2 2 mixtureincubatedincubated of mixturemixture H2O2 and ofof formicHH2O2 andand acid formicformic (the reagent acidacid (the(the mix reagentreagent was stored mixmix waswas two stored weeks two at 4 weeksC) and at shaken 4 °C) and at variousshaken ◦ speedsat various at 0 speedsC to mix at 0 the °C reactants. to mix the Reactions reactants. were ReactionsReactions stopped werewere after stoppedstopped 5 min. Theafterafter maximum 55 min.min. TheThe reaction maximummaximum rate wasreaction achieved rate was by 1200achieved rpm by and 1200 statistical rpm and analysis statisti accordingcal analysis to according the Holm-Sidak to thethe Holm-SidakHolm-Sidak method showed methodmethod no signiﬁcantshowed no difference significant between difference the between rates at 1200the rates rpm at and 1200 1400 rpm rpm. and 1400 rpm.

SinceSince thethe oxidationoxidation of DADS occurs in the organic DADS phase by peracid dissolved in it, varyingvarying the amount of of DADS DADS in in the the reaction reaction mix mix does does notnot not actuallyactually actually affectaffect affect itsits its concentrationconcentration concentration relativerelative relative toto thethe peracid.peracid. Therefore,Therefore, thethe reactionreaction followsfollows aa pseudo-zero-order kinetic with respect to DADS.

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2 2 Furthermore,Molecules 2017 it, was22, 770 observed that with pre-incubation of H O and formic acid a higher rate5 of 14 DADS toconversion the peracid. to allicin Therefore, was achieved the reaction than follows when aall pseudo-zero-order reactants were mixed kinetic at withonce, respect suggesting to DADS. that peracidFurthermore, formation it was observeda rate-limiting that with step. pre-incubation This aspect of will H2O be2 and investigated formic acid ain higher the next rate section.of DADS Furthermore, it was observed that with pre-incubation of H2O2 and formic acid a higher rate of DADS conversion to allicin was achieved than when all reactants were mixed at once, suggesting that conversionIn contrast, to allicin varying was achievedthe concentration than when ofall H2 reactantsO2 affected were the mixed rate of at product once, suggesting formation. that Asperacid shown peracid formation was a rate-limiting step. This aspect will be investigated in the next section. formationin Figure 4 was there a rate-limitingwas a linear relationship step. This aspect between will the be investigatedconcentration in of the H2 nextO2 and section. product formation. In contrast, varying the concentration of H2O2 affected the rate of product formation. As shown Therefore, the reaction follows first-order kinetics with respect to the concentration of H2O2. inIn Figure contrast, 4 there varying was a thelinear concentration relationship between of H2O 2theaffected concentration the rate of of H2 productO2 and product formation. formation. As shown in FigureTherefore,4 there the was reaction a linear follows relationship first-order between kinetics with the concentration respect to the concentration of H 2O2 and of productH2O2. formation. Therefore, the reaction follows ﬁrst-order kinetics with respect to the concentration of H2O2.

Figure 4. The rate of allicin formation in relation to the H2O2 concentration. Micro-scale reactions were Figure 4. The rate of allicin formation in relation to the H2O2 concentration.concentration. Micro- Micro-scalescale reactions reactions were performed with varying H2O2 concentrations. The reactions were stopped after 30 min, when performed withwith varying varying H 2HO2Oconcentrations.2 concentrations. The The reactions reactions were were stopped stopped after 30 after min, 30 when min, turnover when turnover of H2O2 was between 14% (start conc. 2.74 M) and 0.9% (start conc. 0.082 M), the linear ofturnover H O was of H between2O2 was 14%between (start 14% conc. (start 2.74 M)conc. and 2.74 0.9% M) (start and conc.0.9% (start 0.082 M),conc. the 0.082 linear M), progression the linear 2progression2 indicates that neither H2O2 nor DADS were limiting in the reaction. indicatesprogression that indicates neither Hthat2O 2neithernor DADS H2O2 were nor DADS limiting were in the limiting reaction. in the reaction. 2.3. Preformation of Performic Acid 2.3. Preformation of Performic Acid We observed that when H2O2 and formic acid were mixed 3 h before the addition of DADS, a 37% conversion of DADS to allicin2 2 was observed within seconds and that the conversion was >80% We observed that when H 2OO2 andand formic formic acid acid were were mixed mixed 3 3h hbefore before the the addition addition ofof DADS, DADS, a a37% 37% completeconversion conversion after of of120 DADS DADS min (Figureto to allicin allicin 5). was was observed observed wi withinthin seconds seconds and and that that the the conversion was >80% complete after 120 min (Figure(Figure5 5).). 100 90 10080 9070 8060 7050 6040

conversion (%) 5030 4020 10

conversion (%) 30 0 20 0 50 100 10 time (min) 0 0 50 100 Figure 5. Effect of allowing 3 h at 0 °C for the preformatitime (min)on of performic acid on the rate of conversion of DADS to allicin. H2O2 and formic acid were mixed according to the micro-scale reaction procedure and incubated on ice for 3 h before DADS was added. The reactions were stopped with methanol, Figureseparated 5. Effect by of HPLC allowing and quantified 3 h at 0 °C◦C with for for thea the UV preformati preformation detector aton 254 of nm. performic acid on the rate of conversion of DADS to allicin. H2OO22 andand formic formic acid acid were were mixed mixed according according to to the the micro-scale micro-scale reaction reaction procedure

and incubated on ice for 3 hh beforebefore DADSDADS waswas added.added. The reactions were stopped with methanol, separated by HPLC and quantiﬁedquantified withwith aa UVUV detectordetector atat 254254 nm.nm.

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Without preformationpreformation ofof thethe performicperformic acidacid thethe reactionreaction needsneeds ~1.5~1.5 hh toto reachreach >35%>35% conversion and showed >80% conversion only after ~4 h (Figure 11).). ThisThis illustratesillustrates clearlyclearly thatthat thethe formationformation ofof performic acid is rate limiting for allicin synthesis, therefore, we decided to investigate systematically the effect ofof the pre-incubation timetime ofof HH22O2 and formic acid on the conversion rate of DADS to allicin in order toto optimizeoptimize thisthis stepstep inin thethe protocolprotocol (Figure(Figure6 6).).

90 80 70 60 50 40

conversion % 30 RT 20 1 °C 10 0 0 50 100 150 200 250 300 time (min)

Figure 6. 6. TheThe effect effect on onthe thepre-incubation pre-incubation time for time performic for performic acid formation acid formation on the rate on of the DADS rate conversion of DADS

conversionto allicin. H2O to2 and allicin. formic H acid2O2 wereand mixed formic according acid were to th mixede micro-scale according reaction to theprocedure micro-scale and incubated reaction procedureat room temperature and incubated or 1 at°C roomfor the temperature indicated ti ormes 1 ◦beforeC for theDADS indicated was adde timesd. The before reactions DADS were was added.stopped Theafter reactions 5 min bywere addition stopped of methanol, after 5 min separa byted addition by HPLC of methanol, and quantified separated with bya UV HPLC detector and quantiﬁedat 254 nm. A with maximal a UV turnover detector was at 254 reached nm. Aafter maximal 100 min turnover at RT, indicating was reached that maximum after 100 minperformic at RT, indicatingacid formation that maximumwas achieved performic after that acid time. formation was achieved after that time.

We investigatedinvestigated the turnoverturnover of DADSDADS to allicinallicin dependingdepending on the pre-incubation time of the standard amounts of H2O2 and formic acid at 0 °C◦ and at RT. The maximum turnover was reached standard amounts of H2O2 and formic acid at 0 C and at RT. The maximum turnover was reached between 100 and 180 min, followed by decrease of the turnover. Our observations are in accordance with those of Filippis et al. [[17]17] whowho showed,showed, that the formation of performic acid was a slow temperature dependent mechanism. mechanism. In In their their experi experimentalmental setup setup the the maximum maximum turnover turnover of of25% 25% of the H2O2 was reached after 100 min at 30 °C;◦ thereafter the concentration started to decrease due to of the H2O2 was reached after 100 min at 30 C; thereafter the concentration started to decrease due toperformic performic acid acid decomposition. decomposition. Thus Thus,, in our in optimized our optimized allicin synthesis allicin synthesis protocol protocolwe recommend we recommend a 100 min apre-incubation 100 min pre-incubation step at RT stepto pre-form at RT to the pre-form performic the performicacid. acid.

2.4. Influence Inﬂuence of Formic Acid Concentration and Amount of H22O2 onon the the Conversion Conversion of of DADS DADS to to Allicin Allicin Having established that preformation of performicperformic acid greatly enhanced the conversion of DADS to allicin, we we analysed analysed the the proced procedureure with with respect respect to to formic formic acid acid and and H2 HO22O concentrations.2 concentrations. In Inmicro-scale micro-scale reactions reactions higher higher amounts amounts of of acid acid and and H H2O2O2 increased2 increased the the rate rate of of the the reaction reaction (Figure (Figure 77),), but when the reaction volume waswas scaledscaled upup thisthis effecteffect waswas lessless pronounced.pronounced. SeeSee SectionSection 2.52.5..

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120 ratio peroxide to 120 ratioDADS: peroxide to 100 DADS:2.16 fold 100 2.16 fold 1.1 fold 80 1.1 fold 80 60 60

conversion (%) 40

conversion (%) 40 20 20 0 0 35 45 55 65 75 85 35 45 55formic acid 65 (%) 75 85 formic acid (%) Figure 7. The influence of formic acid concentration and amount of H2O2 on the conversion of DADS Figure 7. The influence of formic acid concentration and amount of H2O2 on the conversion of to allicin. In a series of microscale reactions, the amount of H2O2 was varied three times from 1 mol DADSFigure to 7. allicin. The influence In a series of formic of microscale acid concentration reactions, and the amount amount of H of2O H2 on2O 2thewas conversion varied threeof DADS times H2O2 per mol DADS to 2.16 mol H2O2 per mol DADS, indicated with different data point styles. The fromto allicin. 1 mol In H 2aO series2 per of mol micro DADSscale toreactions, 2.16 mol the H amount2O2 per of mol H2O DADS,2 was varied indicated three withtimes different from 1 mol data concentration of formic acid was also varied between 40% and 85% in the reaction. All H2O2-formic pointH2O styles.2 per mol The DADS concentration to 2.16 mol of H formic2O2 per acid mol was DADS, also indicated varied between with different 40% and data 85% point in styles. the reaction. The acid mixtures were pre-incubated for 3 h on ice. The lower black data points (50% formic acid; 47% concentration of formic acid was also varied between 40% and 85% in the reaction. All H2O2-formic All H2O2-formic acid mixtures were pre-incubated for 3 h on ice. The lower black data points (50% conversion) are conform to standard micro-scale reaction conditions. The figure shows that higher formicacid acid;mixtures 47% were conversion) pre-incubated are conform for 3 h to on standard ice. The micro-scale lower black reaction data points conditions. (50% formic The figure acid; 47% shows concentrations of acid and peroxide enhance the speed of the reaction. thatconversion) higher concentrations are conform ofto acidstandard and peroxidemicro-scale enhance reaction the conditions. speed of theThe reaction. figure shows that higher concentrations of acid and peroxide enhance the speed of the reaction. 2.5. Accelerated Allicin Synthesis 2.5. Accelerated Allicin Synthesis 2.5. AcceleratedNot all of Allicin the advantages Synthesis observed by altering parameters in the micro-scale reactions were completelyNotNot all all of oftransferable the the advantages advantages to scaled observed observed up reactions. byby alteringaltering The parametersreaction speed in in thethe was micr micro-scale noto-scale as high reactions reactions as on werethe were completelycompletelymicroscale transferable andtransferable the formation to scaledto scaled of up byproducts reactions.up reactions. became The reactionThe more reaction prevalent speed speed was (data not was not as not high shown). as as high on The the aslatter microscale on arethe andmicroscaleproblems the formation whichand the ofcould formation byproducts be due of lesser byproducts became mixing more inefficibecame prevalentency more and (dataprevalent emulsion not shown).(data formation not Theshown). on latter the Thelarger are latter problems scale. are In order to avoid inadequate mixing, we used methanol to combine the two phases and prevent whichproblems could which be due could lesser be mixingdue lesser inefficiency mixing ineffici andency emulsion and emulsion formation formation on the larger on the scale. larger Inscale. order emulsion formation (Section 3.2.3.). In this way, a conversion of >98.46 ± 0.45% in just 15 min was toIn avoid order inadequate to avoid inadequate mixing, we mixing, used methanolwe used methanol to combine to thecombine two phasesthe two and phases prevent and prevent emulsion achieved (Figure 8). formationemulsion (Section formation 3.2.3 (Section). In this 3.2.3.). way, In a this conversion way, a conversion of >98.46 ±of 0.45%>98.46 in± 0.45% just 15 inmin just was15 min achieved was (Figureachieved8). (Figure 8). 600

600500 [254 nm] 500400 [254 nm] 400300

300200

200100 relative absorbance

1000 relative absorbance 0 5 10 15 20 25 30 35 0 time [min] 0 5 10 15 20 25 30 35

time [min] Figure 8. Chromatogram of crude allicin after synthesis using pre-formed performic acid. The reaction was extracted with dichloromethane (DCM) and the solvent was removed by rotary evaporation. The FigureFigurecrude 8. 8.productChromatogram Chromatogram already showed of of crude crude good allicin allicin purity. afterafter synthesis using pre-formed pre-formed performic performic acid. acid. The The reaction reaction waswas extracted extracted with with dichloromethanedichloromethane (DCM) (DCM) and and the the solvent solvent was was removed removed by rotary by rotary evaporation. evaporation. The

Thecrude crude product product already already showed showed good good purity. purity.

Molecules 2017, 22, 770 8 of 13 Molecules 2017, 22, 770 8 of 14

2.6. Puriﬁcation2.6. Purification of Allicin of Allicin

AfterAfter quenching quenching the the reaction reaction by by addition addition ofof HH2O,O, the the reaction reaction mixture mixture consists consists of allicin, of allicin, DADS, DADS, formic acid, H2O2, and byproducts. The organic compounds were extracted by partitioning against formic acid, H2O2, and byproducts. The organic compounds were extracted by partitioning against eithereither dichloromethane dichloromethane (DCM) (DCM) or or diethyl-ether. diethyl-ether. In the Lawson Lawson method method remaining remaining acetic acetic acid acidwas was removed by washing the organic phase with Na2CO3 solution or extracting several times with water. removed by washing the organic phase with Na CO solution or extracting several times with water. This, however, leads to a loss of allicin, some of2 which3 partitions into the aqueous phase. A further This,advantage however, of leads using to formic a loss acid of allicin,as a catalyst some becomes of which apparent partitions here. intoFormic the acid aqueous is more phase. volatile Athan further advantageacetic acid of using and, therefore, formic acid more as easily a catalyst removable becomes under apparent reduced here. pressure Formic at room acid temperature, is more volatile thus than aceticswitching acid and, to therefore, evaporation more instead easily of washing removable and, under hence, reducedavoiding pressurethe Na2CO at3 washing room temperature, step. thus switchingAfter to evaporation rotary evaporation, instead separating of washing allicin, and, hence,DADS and avoiding byproducts the Na is2 COchallenging,3 washing due step. to the Aftersimilar rotary physical evaporation, properties of separatingthese compounds. allicin, The DADS Lawson and method byproducts partitioned is challenging, repeatedly between due to the similarn-hexane physical and properties water to accumulate of these compounds. allicin in the aqueous The Lawson phase. method The calculated partitioned logP values repeatedly (clogP between) of n-hexaneallicin and (1.35), water DADS to accumulate(2.95), and probable allicin inbyproducts the aqueous such phase.as vinyl-dithiine The calculated (2.69) and log ajoeneP values (1.97) (clog P) of allicin(Chemdraw, (1.35), DADS see Section (2.95), 3) and indicate probable that byproductsallicin is the such least ashydrophobic vinyl-dithiine molecule. (2.69) andNonetheless, ajoene (1.97) repeated extractions lead to further losses of allicin. To circumvent this we used silica gel (Chemdraw, see Section3) indicate that allicin is the least hydrophobic molecule. Nonetheless, repeated chromatography to separate allicin from the other compounds (Figure 9). The structure of the final extractionsproduct lead was toconfirmed further lossesand the of purity allicin. was To determined circumvent by 13 thisC-NMR we used and 1 silicaH-NMR, gel respectively. chromatography to separate allicin from the other compounds (Figure9). The structure of the ﬁnal product was conﬁrmed and the purity was determined by 13C-NMR and 1H-NMR, respectively.

1200

1000 crude (254 nm) 800 purified 600

400

200

0 relative absorbance 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 time (min)

Figure 9. Chromatograms of crude allicin after synthesis using formic acid as a catalyst and rotary Figure 9. Chromatograms of crude allicin after synthesis using formic acid as a catalyst and rotary evaporationevaporation and and afterwards afterwards puriﬁcation purification by bysilica silica gel chromatography. The The crude crude product product contained contained impuritiesimpurities with with retention retention times times of of about about 2 2 min min andand 18 min, min, respectively, respectively, whilst whilst the thepurified puriﬁed product product showedshowed >98% >98% purity. purity. Respresentative Respresentative traces traces of of ourour repeated routine routine syntheses syntheses are are shown. shown.

2.7. Reaction2.7. Reaction Mechanism Mechanism We reasonedWe reasoned that ifthat allicin if allicin synthesis synthesis proceeded proceeded by direct by direct oxidation oxidation of DADS of DADS (1) without (1) without oxidative cleavageoxidative of the cleavage S–S bond, of the then S–S bond, a mixture then a ofmixture DADS of andDADS dipropyl-disulfide and dipropyl-disulfide (DPDS, (DPDS, 2) 2) would would yield yield allicin (DATS, 3) and dipropyl-thiosufinate (propicin, DPTS, 6) only. In contrast, if oxidative allicin (DATS, 3) and dipropyl-thiosufinate (propicin, DPTS, 6) only. In contrast, if oxidative cleavage of the S–S bond occurred, then mixed allyl-propyl thiosulfinates should be further products cleavagebecause of the of S–Srandom bond condensation occurred, then of the mixed respective allyl-propyl sulfenic thiosulfinates acids (Scheme should 2). Thus, be furtherformation products of becauseS-allyl-propane-1-sulfinothioate of random condensation (4) of and the S-propyl-prop-2-ene-1-sulfinoth respective sulfenic acidsioate (Scheme (5) would2). Thus, be predicted. formation of S-allyl-propane-1-sulfinothioate (4) and S-propyl-prop-2-ene-1-sulfinothioate (5) would be predicted. A mixture of DADS (1) and DPDS (2) was oxidized by performic acid, as described in Section 3.2.4. After the reaction was quenched products were extracted with DCM. LC-MS analysis of the crude extracts showed, in addition to single peaks at 6 min and 11 min, which were identified as allicin (3) and propicin (6), respectively, a double peak at 8 min from the mixed thiosulfinates (4,5). Data in Figure 10 are a combination of the UV absorption chromatogram detected by HPLC and the mass signals detected with LC-MS. These data indicate that oxidation of alkyl disulfides to thiosulfinates by peracids proceeds via oxidative cleavage of the S–S bond, but does not formally rule out parallel direct S-atom oxidation without S–S bond cleavage. Therefore, we suggest the reaction mechanism shown in Scheme3. Molecules 2017, 22, 770 9 of 13 Molecules 2017, 22, 770 9 of 14

Scheme 2. The basic reaction for mixed thiosulfinate synthesis from DADS and DPDS by oxidation Scheme 2. The basic reaction for mixed thiosulﬁnate synthesis from DADS and DPDS by oxidation by by peracid generated with H2O2 in the reaction mixture. peracid generated with H2O2 in the reaction mixture. A mixture of DADS (1) and DPDS (2) was oxidized by performic acid, as described in Section 3.2.4. After the reaction was quenched products were extracted with DCM. LC-MS analysis of the crude extracts showed, in addition to single peaks at 6 min and 11 min, which were identified as allicin (3) and propicin (6), respectively, a double peak at 8 min from the mixed thiosulfinates (4,5). Data in Figure 10 are a combination of the UV absorption chromatogram detected by HPLC and the mass signals detected with LC-MS. These data indicate that oxidation of alkyl disulfides to thiosulfinates by peracids proceeds via oxidative cleavage of the S–S bond, but does not formally rule out parallel direct S-atom oxidation without S–S bond cleavage. Therefore, we suggest the reaction mechanism shown in Scheme 3.

Figure 10. Synthesis of mixed thiosulfinates. According to Scheme2 a reaction mechanism via the formation of sulfenic acid would be expected to produce mixed thiosulfinates from a mixture of reacting alkyl disulfides. The chromatogram was obtained by HPLC and quantified with a UV detector at 254 nm. Masses were identified in a separate LC-MS analysis using the same column and gradient. Insets show the m/z ratios in MS-traces and the respective structures of the major ions. The masses obtained fit to the expected molecules: 6 min, 162.92 Da—allicin (3); 8 min, 165.00 Da—S-allyl-propane-1-sulfinothioate (4) and S-propyl-prop-2-ene-1-sulfinothioate (5); 11 min, 166.92 Da—propicin (6). Molecules 2017, 22, 770 10 of 14

Figure 10. Synthesis of mixed thiosulfinates. According to Scheme 2 a reaction mechanism via the formation of sulfenic acid would be expected to produce mixed thiosulfinates from a mixture of reacting alkyl disulfides. The chromatogram was obtained by HPLC and quantified with a UV detector at 254 nm. Masses were identified in a separate LC-MS analysis using the same column and gradient. Insets show the m/z ratios in MS-traces and the respective structures of the major ions. The masses obtained fit to the expected molecules: 6 min, 162.92 Da—allicin (3); 8 min, 165.00 Da—S-allyl- propane-1-sulfinothioate (4) and S-propyl-prop-2-ene-1-sulfinothioate (5); 11 min, 166.92 Da—propicin (6).

Scheme 3. The oxidation of disulfides proceeds via the formation of an allyl cation and an allylsulfenic Scheme 3. The oxidation of disulﬁdes proceeds via the formation of an allyl cation and an allylsulfenic acid in the first step. In a second step the allyl cation reacts with water to form a second allylsulfenic acid in the ﬁrst step. In a second step the allyl cation reacts with water to form a second allylsulfenic acid. The resulting sulfenic acids condense in a third step to form allicin. acid. The resulting sulfenic acids condense in a third step to form allicin. 3. Materials and Methods

53 3-Line-Degasser, a Jasco LG-980-02 ternary gradient unit, a Jasco PU-980 intelligent HPLC pump, a Jasco CO-2060Plus Intelligent column thermostat, a Jasco AS-1555 intelligent sampler, a Jasco UV-2077 multi-wavelength UV-VIS detector, and a Jasco LC-Net II/ADC. Jasco ChromPass Chromatography Data System Version 1.8.6.1 was used for control and analysis (Groß-Umstadt, Germany). Solvent A (H2O) was obtained using a Satorius Stedim Biotech Arium® Pro VF (Goettingen, Germany). Solvent B (methanol (ultra) Gradient HPLC Grade) was purchased from J.T. Baker. (Center Valley, PA, USA). LC-MS was performed using a Bischoff Chromatography Hyperchrome HPLC Column 150 mm × 4.6 mm packed with Prontosil Kromaplus 100-5-C18 5.0 µm in an Agilent 1200 System (Santa Clara, CA, USA). To solvent A 0.1% formic acid (≥98%, p.a.; Carl Roth (Karlsruhe, Germany)) was added. Shaking of the micro-scale reactions was performed using an Eppendorf Thermomixer comfort (Hamburg, Germany) to define 20 °C and a Hettich Benelux MKR 23 (Geldermalsen, The Netherlands) to define 0 °C. Calculation of logP values was performed using ChemDraw Professional 16.0.0.82 (PerkinElmer, Waltham, MA, USA).

3.2. Methods

3.2.1. Distillation of DADS DADS is commercially only available at 80% purity. For further purification we used distillation under reduced pressure. To enhance the efficacy of distillation a Vigreux column (600 mm) was used. The crude DADS was stirred and tempered in an oil bath. The pressure was reduced to approximately

Molecules 2017, 22, 770 10 of 13

3. Materials and Methods

3.1. Materials DADS (≥80%) was purchased from Sigma Aldrich (Munich, Germany). DPDS (98%) was purchased from Sigma Aldrich. Formic acid (≥98%, p.a.) was purchased from Carl Roth (Karlsruhe, Germany). H2O2 (30%) was purchased from Merck (Darmstadt, Germany). Acetic acid (100%, p.a.) was purchased from Carl Roth. TLC was performed using Merck TLC Silica gel 60 F254 with concentration zone. Solvent A (n-hexane ≥99% p.s.) was purchased from Carl Roth. Solvent B (ethyl acetate ≥99.5% p.s.) was purchased from Carl Roth. Liquid chromatography was performed using silica gel 60 (0.04–0.063 mm (230–400 mesh)) purchased from Carl Roth. HPLC was performed using a Bischoff Chromatography Hyperchrome HPLC column 150 mm× 4.6 mm packed with Prontosil Kromaplus 100-5-C18 5.0 µm (Leonberg, Germany) in a Jasco System composed of: a Jasco DG-2080-53 3-Line-Degasser, a Jasco LG-980-02 ternary gradient unit, a Jasco PU-980 intelligent HPLC pump, a Jasco CO-2060Plus Intelligent column thermostat, a Jasco AS-1555 intelligent sampler, a Jasco UV-2077 multi-wavelength UV-VIS detector, and a Jasco LC-Net II/ADC. Jasco ChromPass Chromatography Data System Version 1.8.6.1 was used for control and analysis (Groß-Umstadt, ® Germany). Solvent A (H2O) was obtained using a Satorius Stedim Biotech Arium Pro VF (Goettingen, Germany). Solvent B (methanol (ultra) Gradient HPLC Grade) was purchased from J.T. Baker. (Center Valley, PA, USA). LC-MS was performed using a Bischoff Chromatography Hyperchrome HPLC Column 150 mm × 4.6 mm packed with Prontosil Kromaplus 100-5-C18 5.0 µm in an Agilent 1200 System (Santa Clara, CA, USA). To solvent A 0.1% formic acid (≥98%, p.a.; Carl Roth (Karlsruhe, Germany)) was added. Shaking of the micro-scale reactions was performed using an Eppendorf Thermomixer comfort (Hamburg, Germany) to deﬁne 20 ◦C and a Hettich Benelux MKR 23 (Geldermalsen, The Netherlands) to deﬁne 0 ◦C. Calculation of logP values was performed using ChemDraw Professional 16.0.0.82 (PerkinElmer, Waltham, MA, USA).

3.2. Methods

3.2.1. Distillation of DADS DADS is commercially only available at 80% purity. For further puriﬁcation we used distillation under reduced pressure. To enhance the efﬁcacy of distillation a Vigreux column (600 mm) was used. The crude DADS was stirred and tempered in an oil bath. The pressure was reduced to approximately 50 mbar. At an oil bath temperature of 120 ◦C the DADS fraction evaporated. The boiling point under these conditions was 80.5 ◦C. A purity of 98% was determined by HPLC.

3.2.2. Synthesis of Allicin without Pre-Formed Performic Acid Distilled diallyl disulﬁde (DADS; 2 g, 13.7 mmol) was mixed in 5 mL formic acid and stirred for ◦ 5 min at 0 C. H2O2 (30%; 3 mL, 29.6 mmol) was added slowly to the mixture. The reaction was stopped after approximately 4 h by addition of 25 mL distilled water and the mixture was extracted three times with DCM. The solvent was removed under reduced pressure and the product was dissolved in the eluent, a mixture of n-hexane and ethyl-acetate (2:1). Separation was performed via liquid chromatography using 150 mm silica gel 60 in a column with a diameter of 30 mm. Fractions were collected into tubes cooled in an ice bath and TLC was used to identify fractions containing solely allicin. Those fractions were combined, dried over amorphous anhydrous sulfate (e.g., MgSO4 or CaSO4) and ﬁltered. The solvents were removed under reduced pressure at RT to yield a clear, oily substance that smells like garlic. Yield: 1.64 g, 10.1 mmol, 74%. 1 13 H-NMR (500 MHz, CDCl3): δ3.70–3.75 (m, 4H); 5.14–5.42 (m, 4H); 5.68–5.88 (m, 2H); C-NMR: (125 MHz, CDCl3) δ35.08, 59.82, 119.11, 124.10, 125.78, 132.8. Molecules 2017, 22, 770 11 of 13

3.2.3. Synthesis of Allicin Using Pre-Formed Performic Acid Distilled diallyl-disulﬁde (DADS; 0.5 g, 3.5 mmol) was mixed in 2.5 mL methanol and stirred for 5 min at 0 ◦C. Performic acid solution (2.0 mL) (as described in Section 3.2.6.) was added slowly to the mixture. The reaction was quenched after 15 min by addition of 25 mL distilled water and the mixture was extracted three times with DCM. The solvent was removed under reduced pressure and the product was dissolved in a mixture of n-hexane and ethyl-acetate (2:1). Separation was performed via liquid chromatography using 150 mm silica gel 60 in a column with a diameter of 30 mm and n-hexane and ethyl acetate (2:1) as eluent. Fractions were collected into tubes cooled in an ice bath and TLC was used to identify fractions solely containing allicin. Those fractions were combined, dried over an anhydrous sulfate, and ﬁltered. The solvents were removed under reduced pressure at RT to yield a clear, oily substance that smells like garlic. Yield: 0.52 g, 3.204 mmol, 92%. 1 13 H-NMR (500 MHz, CDCl3): δ 3.70–3.75 (m, 4H); 5.14–5.42 (m, 4H); 5.68–5.88 (m, 2H); C-NMR (125 MHz, CDCl3): δ35.08, 59.82, 119.11, 124.10, 125.78, 132.8.

3.2.4. Synthesis of Mixed Thiosulfinates Diallyl disulfide (DADS; 1 g, 6.84 mmol) and dipropyl disulfide (DPDS; 1g, 6.65 mmol) were ◦ mixed in 5 mL formic acid and stirred for 5 min at 0 C. H2O2 (30%; 3 mL, 29.6 mmol) was added slowly to the mixture. The reaction was quenched after approximately 4 h by addition of 25 mL distilled water and the mixture was extracted three times with DCM. The solvent was removed under reduced pressure and the crude products were analysed by HPLC and HPLC-MS.

3.2.5. Micro-Scale Reaction DADS (10 mg, 68.4 µmol) was mixed in 25 µL of either formic or acetic acid in a 2.0 mL reaction tube on ice. The formic acid-containing tubes were placed in a cooling shaker at 0 ◦C, the acetic ◦ acid-containing tubes were placed in a shaker at 20 C. Then H2O2 solution (30%, 15 µL, 148 µmol) was added to the mixture and the reaction was initiated by shaking at 1200 rpm. For sample collection, single tubes were removed and the reaction was quenched by diluting the mixture to 2 mL with methanol. The samples were stored at −20 ◦C prior to HPLC analysis.

3.2.6. Performic acid Pre-Formation

If not explained differently, H2O2 and formic acid were mixed (in a ratio of 3:5) and incubated at RT for 90 min. In micro-scale reactions, for instance, 40 µL of that mixture was used instead of adding 25 µL formic acid and 15 µLH2O2.

3.2.7. High-Performance Liquid Chromatography (HPLC) Analysis Reaction mixtures were analyzed by loading each 20 µL sample onto the HPLC. Separation was performed using H2O as mobile phase A and methanol as mobile phase B with the following gradient: 56% A (pre-run); 53% A (10 min); 7% A (15 min); 7%A (30 min); 56% A (31 min); 56% A (35 min) at a ﬂow rate of 1 mL/min and a column thermostat temperature of 25 ◦C. Under these conditions retention times were 4.8 min for allicin and 18.2 min for DADS. Byproducts appearing at 14.9 min and 17.7 min due to their calculated logP values are assumed to be forms of ajoene and vinyldithiine, but were not investigated further at this stage. To quantify allicin and DADS, external standards were used.

3.2.8. Liquid Chromatography-Mass Spectrometry (LC-MS) The LC-MS protocol used the same gradient and column as the HPLC protocol, except for the use of 0.1% formic acid, which was used instead of pure water. The following source conditions were employed: heater—350 ◦C; sheath gas flow rate machine settings (without units)—30; auxiliary Molecules 2017, 22, 770 12 of 13 gas flow rate—5; sweep gas flow rate—0; ion spray voltage—400 kV; capillary temperature—250 ◦C; capillary voltage—82,5 V; tube lens—120 V in a ThermoFischer LTQ XL (Waltham, MA, USA).

3.2.9. Thin Layer Chromatography (TLC) Approximately 2 µL of the reaction mixture was loaded on a silica plate. After drying, the substances were separated using n-hexane/ethyl-acetate mixture (in a ratio of 2:1) as mobile phase. Under these conditions spots were visible under UV light (254 nm). Allicin’s Rf value was 0.70 and DADS’s Rf value was 0.95.

4. Conclusions Our data provide evidence that the reaction mechanism underlying the conversion of DADS to allicin in the presence of formic acid and H2O2 is similar to that already proposed by Nikolic, but without the need for hydroxyl radicals. The unpaired electrons in such radicals might delocalize and would surely result in a number of additional side products for which we see no evidence. As the four possible products from the mixture of disulfides (DPDS and DADS) were formed in approximately equal amounts, we surmise that direct oxidation of the disulfides without chain cleavage is probably not quantitatively significant and, thus, we suggest an oxidative cleavage mechanism for the reaction as shown in Scheme3. We also show that the optimized method we describe here to synthesize allicin is an improvement on the previously-published procedures based on the one of Lawson [10]. Not only does the utilization of formic acid as a catalyst lead to a purer product, since the formation of by-products is decreased, the reaction also occurs faster and is easier to perform under standard conditions. Formic acid offers another advantage during the purification of the product allicin because it is more volatile than acetic acid and therefore easily removed under reduced pressure. Other peroxy-acids such as magnesium monoperoxyphthalate, or chloroperbenzoic acid have also been used [11–13]. In light of economical reasoning, however, the price of formic acid compared to aromatic peroxy-acids is just another argument, which points to formic acid as the catalyst of choice for the synthesis of allicin. Additionally, formic acid, as a naturally-occurring organic molecule produced, for example, by red ants and stinging nettles, is more eco-friendly than most alternatives (with the possible exception of acetic acid) and certainly ‘greener’ than the aromatic alternatives. The use of silica gel chromatography offers the advantage whereby a separation of the product and byproducts can be achieved without further diluting the allicin excessively. Therefore, it is possible to continue the reaction until a maximal turnover is reached, purify the crude product, and obtain pure allicin rather easily. A suggested optimized protocol for the synthesis of allicin, taking into account the various individual improvements we describe here are, therefore, as follows:

1. Use redistilled DADS and add methanol to combine the aqueous and organic phases (see Section 3.2.3.) or keep the final reaction volume small to promote efficient mixing and achieve a high conversion rate. 2. Use formic acid as the acid catalyst and pre-form performic acid as described in Section 3.2.6. 3. Cool the reagents and carry out the reaction on ice. 4. Slowly add performic acid solution. 5. Continually stir the reactants as efficiently as possible and carry out the reaction at 0 ◦C for just 15 min. 6. Quench the reaction with water.

Acknowledgments: F.A. and A.J.S. gratefully acknowledge ﬁnancial support from RFwN and RWTH Aachen University. R.L. gratefully acknowledges the ﬁnancial support from University of the Saarland, Saarbruecken, and RWTH Aachen University. The NMR laboratories in the chemistry departments of the RWTH Aachen University (Ines Bachmann-Remy) and Saarbruecken University are thanked for providing NMR facilities and Kilian Smith for the use of the LC-MS facilities in the Institute of Ecotoxicology, RWTH Aachen University. Molecules 2017, 22, 770 13 of 13

Author Contributions: F.A. and R.L. contributed equally to the practical work and writing the manuscript. A.J.S. provided supervision and wrote and edited the manuscript. C.J. edited the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Sample Availability: Samples of the compounds are not available from the authors.

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