research article enzymatic synthesis of sorboyl-polydatin...

Research ArticleEnzymatic Synthesis of Sorboyl-Polydatin Prodrugin Biomass-Derived 2-Methyltetrahydrofuran and AntiradicalActivity of the Unsaturated Acylated Derivatives

Zhaoyu Wang1 Yanhong Bi1 Rongling Yang1 Xiangjie Zhao1 Ling Jiang2 Chun Zhu1

Yuping Zhao1 and Jianbo Jia1

1School of Life Science and Food Engineering Huaiyin Institute of Technology Huairsquoan 223003 China2College of Food Science and Light Industry Nanjing Tech University Nanjing 211816 China

Correspondence should be addressed to Zhaoyu Wang biowzy126com and Yanhong Bi byhfood126com

Received 31 May 2016 Revised 3 August 2016 Accepted 7 August 2016

Academic Editor Pengjun Shi

Copyright copy 2016 Zhaoyu Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Efficient and highly regioselective synthesis of the potential 610158401015840-O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahy-drofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time Under the optimal conditions the initialreaction rate maximum substrate conversion and 610158401015840-regioselectivity were as high as 865mMh 100 and 100 respectivelyKinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF comparedto the traditional organic solvents With respect to the antioxidant properties three unsaturated ester derivatives showed slightlylower DPPH radical scavenging activities than the parent agent Interestingly further studies also revealed that the antiradicalcapacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11 The reason mightbe attributed to the increased steric hindrance derived from the acyl residues in derivatives

1 Introduction

Polydatin (also known as piceid and resveratrol-3-O-120573-mono-D-glucoside) is the key pharmacodynamic constituentfrom Polygonum cuspidatum Sieb et Zucc which is an exten-sively used traditional Chinese medicinal plant in the clinicaltreatment of platelet aggregation diabetes mellitus ische-miandashreperfusion injuries and burn-generated cardiac dys-function and so forth [1 2] Nowadays the highly lipophilicdrugs are becoming more and more prevalent and welldesignedwith enhanced pharmacological activity in pharma-ceutical industry [3 4] However similar to other originalnatural drugs polydatin generally suffers from poor solu-bility low bioavailability and unsatisfactory stability in lipidformulations because of its polyhydroxy structure [5] Con-sequently in order to overcome the aforementioned disad-vantages and improve the bioavailability in lipid systems oneof the most effective strategies is to synthesize ester prodrugsof polydatin For example structure-function relationships

revealed that the acylated derivatives of rutin pruningand naringin presented improved biological activities andphysicochemical properties compared to the correspond-ing parental agents [6 7] Moreover further investigationsalso found that natural compounds modified by 120572- or 120573-unsaturated groups usually displayed the higher biologicalactivity compared with the parent agents [8 9]

Chemically the acyl chlorides and anhydrides are usuallyrequired as acylating reagents for acylating flavonoids whichare characterized by the poor selectivity tedious protection-unprotection steps and low product yields [10] Fortunatelyenzymatic strategy instead of conventional chemical methodhas beenwell established for acylating compounds possessingsimilar reactive hydroxyl groups [11] Recently our researchgroup has successfully explored an efficient and practicalenzymatic route for the regioselective acylation of polyhy-droxy nucleosides by using medium engineering enzymeengineering and substrate engineering strategies with excel-lent regioselectivities and conversions [12 13]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4357052 7 pageshttpdxdoiorg10115520164357052

2 BioMed Research International

OHOHO

HO

O

OH OHHO

HO

HO

OH

OH

OH

OO

OO

Polydatin

Vinyl sorbate

5 5

49984006

998400998400

4998400998400

3998400998400

49984006

998400998400

4998400998400

39984009984002

998400998400

CALB 2-MeTHF 60∘C

6998400998400-O-Sorboyl-polydatin

1998400998400998400

2998400998400

Scheme 1 CALB-catalyzed acylation of polydatin with vinyl sorbate

Over the past several years biobased solvents that isgreen solvents derived from renewable raw materials havearoused great interest as environmentally friendly alterna-tives to traditional organic solvents in catalysis and organicchemistry particularly in biocatalytic reactions with promis-ing physicochemical properties of renewability biodegrad-ability environmental benignancy stability and so forth [14]Among these specific solvents 2-MeTHF produced fromrenewable resources of lignocellulosic biomass such as corncrops bagasse and oat hulls exerts clear advantages like thelow miscibility with water high boiling point (82∘C) neg-ligible toxicity higher stability and abiotic degradability byexposure to sunlight and air [14 15] Following the pioneeringwork of Simeo and coworkers in enzymatic acylation ofnucleosides in 2-MeTHF [16] biocatalysts have shown higheractivity and stability in this ecofriendly medium than intraditional organic solvents

Therefore in this study an effective enzymatic method in2-MeTHF is explored for preparing potential dual prodrugof sorbic acid (an unsaturated fatty acid with noticeablebactericidal and fungicidal properties) ester of polydatincatalyzed by Candida antarctica lipase B (CALB) a versatileenzyme immobilized on macroporous acrylic resin with amolecular weight of 33 kDa and approximate dimensions of10 A times 4 A times 12 A [17 18] (Scheme 1) The kinetic study of theacylation was explored in detail to better identify the effectof 2-MeTHF on the performance of the enzyme Particularlythe free radical scavenging activities of several unsaturatedacylated derivatives were also preliminarily studied

2 Materials and Methods

21 Materials CALB (Candida antarctica lipase B immobi-lized on macroporous acrylic resin 10000Ug) was fromNovozymes Co Ltd China Vinyl sorbate vinyl undeceno-ate and vinyl crotonate were obtained from TCI Poly-datin 22-diphenyl-1-picrylhydrazyl (DPPH) 2-MeTHF andt-amyl alcohol were purchased from Sigma-Aldrich All otherreagents were of analytical grade and were dried by 4 Amolecular sieves

22 Enzymatic Acylation Procedure The acylation wasconducted with polydatin (003mmol) vinyl sorbate(027mmol) anhydrous solvent (3mL) and 100mg lipasewhile stirring at 200 rpm and at a certain temperature To

determine the concentrations of the polydatin and regiose-lectivities of the acylated products aliquot fractions (50 120583L)were withdrawn at specified time intervals from the reactionsystem followed by a HPLC analysis All experiments wereconducted at least in triplicate and the errors did not exceed5 No chemical acylation was detectable as confirmed bythe control experiments

23 Operational Stability of CALB After the maximum sub-strate conversion reached during the batch acylation of poly-datin the immobilized enzyme was separated by filtrationand washed three times with 2-MeTHF or THF Then thereused prepared catalyst was added into the fresh reactionmixture (3mL) containing 003mmol polydatin 045mmolvinyl crotonate and 100mg CALB at 60∘C and 200 rpmfollowed by the assay of its activity and maximum polydatinconversion The initial activity and maximum conversionobtained in the first batchwere set to 100The residual activitywas defined as the ratio of the reused enzyme activity relativeto the original enzyme activity in the same reaction system

24 Determination of the Enzymatic Kinetic Constants Thekinetic constants of enzymatic sorboylation were investi-gated at different polydatin concentrations in 2-MeTHF (2ndash14mM) THF (5ndash50mM) acetone (4ndash16mM) and t-butanol(2ndash18mM) The reactions were carried out in 3mL solventcontaining different polydatin concentration 9 equiv of vinylsorbate and 100mg CALB at 60∘C and 200 rpm The kineticconstants (119870

119898and 119881max) were calculated from Hanes-Woolf

plots

25 Determination of the Enzymatic Apparent ActivationEnergy (119864

119886) The apparent activation energy values (119864

119886) of

enzymatic acylation in 2-MeTHF THF acetone and t-butanol were investigated Polydatin (003mmol) CALB(100mg) and vinyl sorbate (027mmol) were added intothe anhydrous solvent (3mL) and incubated at differenttemperatures (in the range of 35ndash50∘C) and 200 rpm119864

119886value

of enzymatic sorboylation was calculated according to thelinear regression analysis of the Arrhenius plot

26 NMR and HPLC Analytical Procedure Qualitative anal-ysis of the reaction mixtures was conducted by HPLC usinga 46mm times 250mm (5 120583m) Eclipse Plus-C18 column (AgilentTechnologies Industries Co Ltd USA) a UV detector at

BioMed Research International 3

Table 1 Effect of solvent on CALB-catalyzed acylation of polydatin with vinyl sorbatea

Medium log119875 Viscosityb Solubility (mM)c 1198810(mMh) 119862 () 610158401015840-Regioselectivity ()

2-MeTHF 099 060 851 plusmn 042 304 plusmn 015 9957 plusmn 003 100t-Amyl alcohol 124 370 587 plusmn 021 240 plusmn 011 7931 plusmn 071 100t-Butanol 060 330 654 plusmn 026 164 plusmn 005 9900 plusmn 002 100Cyclohexanone 143 220 1396 plusmn 039 083 plusmn 006 6672 plusmn 101 100Acetone minus023 032 1263 plusmn 053 062 plusmn 003 9900 plusmn 002 100THF 049 055 7217 plusmn 298 035 plusmn 001 4322 plusmn 176 100Dioxane minus110 130 2072 plusmn 101 021 plusmn 001 2516 plusmn 131 100aThe reactions were carried out at 40∘C and 200 rpm by adding 003mmol polydatin 015mmol vinyl sorbate and 100mg CALB into 3mL anhydrous solventbThe viscosity of solvents at 25∘CcThe solubility of polydatin was determined by HPLC analyses of the saturated solutions at 25∘C

275 nm and a solvent mixture system of water and methanol(4060 vv) at a flow rate of 10mLmin The retention timesfor polydatin and 610158401015840-ester were 259 and 527min respec-tively The acylated derivative was purified by flash columnchromatography using ethyl acetate (EA)petroleum ether(PE) and its chemical structure was determined by 13C NMR(100MHz) and 1HNMR (400MHz) (Bruker DRX-400NMRSpectrometer Bruker Co Germany) in DMSO-119889

6

610158401015840-O-Sorboyl-polydatin 1H NMR (DMSO-1198896) 120575 963 (br s

1H OH phenolic) 951 (br s 1H OH phenolic) 743 (d 2H119869 = 60Hz H

21015840 + H61015840) 718ndash713 (m 1H 119869 = 180Hz H

4101584010158401015840)

705 (d 1H 119869 = 108Hz Hvinyl-1) 691 (d 1H 119869 = 108 HzH3101584010158401015840) 688 (t 1H 119869 = 24Hz H

2101584010158401015840) 687 (t 1H Hvinyl-2) 681

(d 1H 119869 = 56Hz H31015840) 670 (br s 1H H

51015840) 663 (br s 1H

H2) 636 (t 1H 119869 = 16Hz H

6) 616ndash612 (m 1H H

5101584010158401015840)

580 (dd 1H 119869 = 12 12Hz H4) 542 (br s 1H OH

210158401015840)

536 (br s 1H OH310158401015840) 524 (br s 1H OH

410158401015840) 494 (d 1H

119869 = 52Hz H110158401015840) 447 (d 1H 119869 = 72 H

610158401015840-1) 411 (dd

1H 119869 = 52 52Hz H610158401015840-2) 374ndash371 (m 1H H

510158401015840) 339ndash

336 (m 1H H210158401015840) 332ndash330 (m 1H H

310158401015840) 325ndash322 (m 1H

H410158401015840) 171 (d 3H 119869 = 44Hz H

6101584010158401015840) 13C NMR (DMSO-119889

6)

120575 16677 (C1101584010158401015840) 15905 (C

3) 15886 (C

5) 15785 (C

41015840) 14564

(C3101584010158401015840) 14043 (C

4101584010158401015840) 13982 (C

1) 12986 (C

21015840 + C61015840) 12895

(C11015840) 12840 (Cvinyl-1) 12576 (Cvinyl-2) 11875 (C

2101584010158401015840) 11605

(C31015840 +C51015840) 10762 (C

6) 10479 (C

2) 10337 (C

110158401015840) 10055 (C

4)

7689 (C210158401015840) 7429 (C

510158401015840) 7362 (C

310158401015840) 7059 (C

410158401015840) 6417 (C

610158401015840)

6024 (C5101584010158401015840) 1877 (C

6101584010158401015840)

27 DPPH Scavenging Activity The antioxidant activity ofpolydatin and its acylated derivatives was examined accord-ing to the previously reported procedure with a few modifi-cations [19] Briefly 2mL of freshly made DPPH solution wasadded into ethanol solutions of individual tested compoundsto start the reaction at a final concentration of 200120583MDPPHEach compound concentration was fixed at 01 02 0408 and 12mgmL respectively The absorbance at 517 nmwas measured against a blank of pure ethanol in a lightproof reaction vessel at room temperature for 30min DPPHscavenging capacity was estimated based on the difference inabsorbance and expressed by the remaining radical DPPH

percentage Triplicate reactions were carried out for eachantioxidant experiment and the results were based on theaverage values

3 Results and Discussions

31 Selection of Suitable Reaction Media The search for suit-able solvent capable of maintaining enzyme activity improv-ing enzyme specificity and accelerating the enzymatic reac-tion process has always been a central problem for bio-chemists [20 21] So far no empirical regularities could befollowed to guide the rational choice of the solvent systemin nonaqueous enzymology The solvent physicochemicalproperties such as the log119875 dielectric constant viscosity dis-solving capacity and molecular size unpredictably affect thebehavior of the enzyme Hence eight organic solvents withdifferent log119875 values (minus110ndash143) were selected to furtherevaluate their effects on the CALB-mediated sorboylation ofpolydatin

As shown in Table 1 the initial reaction rate andconversion of the biocatalytic sorboylation varied greatlywith the use of different solvents For the regioselectivityit is interesting to note that the solvents investigated gaveexclusively acylation at the 610158401015840-hydroxyl group Howeverthere was not an inevitable correlation between the enzymeactivity and solvent properties including the log119875 viscosityand substrate solubility in this acylation In those solventssuch as THF dioxane cyclohexanone and t-amyl alcoholCALB displayed poor to good activities with 2516ndash7931maximal conversions By contrast acetone and t-butanolseemed to be selectable media and gave 9900 substrateconversions but the initial rates were far from satisfactory(062ndash164mMh) However when the reaction was carriedout in ecofriendly 2-MeTHF CALB showed highest reactionrate (304mMh) and excellent conversion (9957) indicat-ing that the biomass-derived 2-MeTHF exhibited prominentbiocompatibility with the lipase compared to the otherconventional organic media Furthermore compared withthe acetone and t-butanol 2-MeTHF may be more beneficialto maintain enzyme activity owing to its more hydrophobiccharacter [22]


2

3

4

5

6In

itial

reac

tion

rate

(mM

h)

Initial reaction rateMaximum conv6998400998400-Regioselectivity

20

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)

146 8 10 1242Vinyl sorbatepolydatin (molar ratio)

(a)


40 45 50 55 60 65 7035Temperature (∘C)

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)

2

4

6

8

Initi

al re

actio

n ra

te (m

Mh

)

(b)

Maximum conv6998400998400-Regioselectivity

20

40

60

80

100

Max

imum

conv

(

)

80 120 160 200 24040Reaction time (min)

20

40

60

80

100

Regi

osele

ctiv

ity (

)

(c)

Figure 1 Effect of the molar ratio of vinyl sorbate to polydatin (a) temperature (b) and reaction time (c) on CALB-catalyzed acylationof polydatin Reaction conditions (a) 003mmol polydatin 100 mg CALB 3mL anhydrous 2-MeTHF 200 rpm various amounts of vinylsorbate 40∘C (b) 003mmol polydatin 100 mg CALB 3mL anhydrous 2-MeTHF 200 rpm 027mmol vinyl sorbate different temperaturesfrom 35 to 70∘C (c) 003mmol polydatin 100 mg CALB 3mL anhydrous 2-MeTHF 200 rpm 60∘C reaction time from 20 to 250min

32 Effect of the SubstrateMolar Ratio Temperature andReac-tion Time The influence of some crucial factors such as thesubstrate molar ratio temperature and reaction time wasalso investigated in detail As can be seen in Figure 1(a) theoptimal molar ratio of vinyl sorbate to polydatin was shownto be 9 suggesting that an excessive amount of vinyl sorbate isrequired due to the reversibility of the reaction Temperaturehas a considerable effect on the conformational unfolding ofthe protein and the reaction equilibrium as well Figure 1(b)illustrates that there was 19 times increase in the initial ratewhen the temperature was raised from 35 to 60∘C beyondwhich further increasing temperature brought a slight dropin the initial reaction rate and maximum conversion Lipase

deactivation caused by high temperature may be one of thereasonable explanations for this phenomenon

Gardossi et al recently demonstrated that it is very neces-sary to monitor the procedure parameters (such as the reac-tion rate selectivity and yield) at a single timepoint [23]So the time course of producing 610158401015840-O-sorboyl-polydatin wasfollowed under the optimal conditions for better understand-ing the enzymatic process It was found in Figure 1(c) thatthe rate of substrate conversion underwent a steep incrementwithin 80min and then a smooth rise with prolonging theincubation time up to 160min This phenomenon may occurbecause the acetaldehyde and sorbic acid from the vinylsorbate partially inactivated the biocatalyst [24 25] After


2-MeTHFTHF

0

20

40

60

80

100

120Re

lativ

e act

ivity

()

2 3 4 5 6 7 8 9 10 11 121Batch

(a)

0

20

40

60

80

100

120

Relat

ive m

axim

um co

nv (

)

2-MeTHFTHF

2 3 4 5 6 7 8 9 10 11 121Batch

(b)

Figure 2 Operational stability of CALB in 2-MeTHF and THF The reactions were carried out at 60∘C and 200 rpm by adding 003mmolpolydatin 027mmol vinyl sorbate and 100mg CALB into 3mL anhydrous 2-MeTHF

reaching a maximum (200min) the substrate conversiongradually dropped with extended reaction time which isin accordance with our previous studies and means thatthe acylated product was enzymatically hydrolyzed duringsubsequent reaction process [26]

33 Operational Stability of the CALB The successful incor-poration of biocatalysts into fine chemicals productionrequires not only high catalytic activities but also excellentoperational stabilities The results for repeated uses of theenzyme in 2-MeTHF and THF are presented in Figure 2 andthe differences are evident The catalytic activity of CALBdecreased only slightly during the first two batches in 2-MeTHF with around 8927 of its original activity beingretained after the second batch whereas only about 4873was obtained in THF In particular after eight successivecycles of reuse CALB still displayed 29 times the residualactivity (7634 versus 2600) in 2-MeTHF compared to inTHF Similarly for themaximal conversion of the operationalstability the catalyst remained 9324 and 1321 respec-tively of their initial maximal conversions in ecofriendly2-MeTHF and traditional THF after being used repeatedlyfor 12 batches showing that the biomass-derived 2-MeTHFexhibited a great potential as the reaction medium for theenzyme-catalyzed acylation

34 Kinetic Studies For better understanding the superiorityof 2-MeTHF measurement of kinetic constants of enzymaticsorboylation by using linear Hanes-Woolf plots was suc-cessfully explored As illustrated in Table 2 the apparentMichaelis constant 119870

119898value of the enzyme in 2-MeTHF

(279mM) is much lower than those attained in t-butanolacetone and THF (1003 1032 and 1072mM resp) Simul-taneously the catalytic efficiency of CALB varied greatlydepending on the nature of media For instance CALB

Table 2 Effect of polydatin concentration on enzymatic acylationin various solventsa

Medium 119881max (mMh) 119870119898(mM) 119881max119870119898 (h

minus1)2-MeTHF 312 279 112tert-Butanol 308 1003 041Acetone 247 1032 024THF 233 1072 022aThe reactions were carried out at 60∘C and 200 rpm by adding variousamounts of polydatin vinyl sorbate (9 equiv) and 100mg CALB into 3mLanhydrous solvent

afforded the highest 119881max119870119898 value of 112 hminus1 in 2-MeTHFsystem which is 37- to 55-fold higher than that in othersolvents studiedThese promising observations indicated thatthe enzyme exerted an extremely higher affinity for thesubstrate and catalytic efficiency in this novel 2-MeTHF ascompared to normal organic solvents

The apparent activation energy (119864119886) for the reaction

is also determined by plotting the data in an Arrheniusplot (Figure 3) The obtained 119864

119886value of 433 KJmol in 2-

MeTHF is lower than that of the sorboylation in other mediaused (507ndash549 KJmol) suggesting that the application of 2-MeTHF has proved to be more beneficial for overcomingthe reaction energy barrier and rendering the process ofenzymatic biotransformation

35 Radical DPPH Scavenging Activity Based on the opti-mization of the enzymatic sorboylation 610158401015840-O-crotonyl- and610158401015840-O-undecenoyl-polydatin were also successfully synthe-sized for better evaluation of the free radical scavengingactivity of the unsaturated polydatin ester derivatives Asdepicted in Figure 4 all of the tested compounds displayedthe obvious dosage-effect relationship on DPPH radicalquenching capacity and the higher concentrations were more


Solvent

549

527

507

THFAcetonet-Butanol2-MeTHF 433

Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)

Figure 3 Arrhenius plots of CALB-catalyzed acylation of polydatinin variousmediaThe reactions were carried out at different temper-atures and 200 rpmby adding 003mmol polydatin 027mmol vinylsorbate and 100mg CALB into 3mL anhydrous 2-MeTHF

effective in quenching free radicals When the concentrationwas fixed at 12mgmL for instance all the unsaturatedacylated products exhibited superior antioxidant activity(945ndash964) However the scavenging activities of threeacylates were always lower than those of the correspondingconcentrations of the parent agent This is in agreement withthe previous findings on the acylated flavonoids [27 28] Forthe effect of the aliphatic acid chain length the unexpectedgradual decreases of the free radical scavenging activitieswere obtainedwith the increment in the unsaturated aliphaticchain length fromC4 toC11 One of themost possible reasonsmight be that the increased steric hindrance derived from theacyl residues in derivatives prevents the substrate moleculefrom smoothly entering radical active sites and thus reducingthe DPPH scavenging activity

4 Conclusions

In this paper a practical enzymatic approach for acylatingpolydatin with vinyl sorbate in biomass-derived 2-MeTHFis described for the first time CALB lipase was used suc-cessfully to acylate polydatin at the 610158401015840-position Detailedinvestigations have revealed that the enzyme in 2-MeTHFgave the lowest119870

119898and 119864

119886values highest119881max and excellent

operational stability which suggested that this ecofriendlysolvent could render the enzyme much higher affinity for thesubstrate and catalytic efficiency For the antioxidant activityintroducing the acyl group into the polydatin would result inthe slight reduction of its DPPH radical scavenging capacityIn addition more detailed physicochemical investigations onthe pH stability 1-octanol-water partition coefficient (log119875)

60

70

80

90

100

Scav

engi

ng ac

tivity

()

04 06 08 10 1202Concentration of antioxidant (mgmL)

Polydatin


6998400998400-O-Crotonyl-polydatin

6998400998400-O-Undecenoyl-polydatin

Figure 4 Comparison of radical DPPH scavenging capacity

apoptosis-inducing capability and so forth of the acylatedproducts are currently in process and will be reported in duecourse

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Authorsrsquo Contributions

Zhaoyu Wang and Yanhong Bi have made equal contribu-tions

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (21676114 31501421) Qing Lan Projectof Jiangsu Province Key Research Program of Industry andInformation Technology of Huairsquoan (HAG2015031) NaturalScience Research Project of Higher Education of JiangsuProvince (16KJB530001) Natural Science Foundation ofJiangsu Province (BK2012243) and Foundation of HuaiyinInstitute of Technology (HGB1401)

References

[1] J Hao C Chen K Huang et al ldquoPolydatin improves glu-cose and lipid metabolism in experimental diabetes throughactivating the Akt signaling pathwayrdquo European Journal ofPharmacology vol 745 pp 152ndash165 2014

[2] W Peng R Qin X Li and H Zhou ldquoBotany phytochemistrypharmacology and potential application of Polygonum cuspida-tum Siebet Zucc a reviewrdquo Journal of Ethnopharmacology vol148 no 3 pp 729ndash745 2013


[3] H Mu R Holm and A Mullertz ldquoLipid-based formulationsfor oral administration of poorly water-soluble drugsrdquo Interna-tional Journal of Pharmaceutics vol 453 no 1 pp 215ndash224 2013

[4] C J H Porter N L Trevaskis and W N Charman ldquoLipidsand lipid-based formulations optimizing the oral delivery oflipophilic drugsrdquo Nature Reviews Drug Discovery vol 6 no 3pp 231ndash248 2007

[5] L Lin B Ni H Lin et al ldquoTraditional usages botanyphytochemistry pharmacology and toxicology of Polygonummultiflorum Thunb a reviewrdquo Journal of Ethnopharmacologyvol 159 pp 158ndash183 2015

[6] G Celiz and M Daz ldquoBiocatalytic preparation of alkyl estersof citrus flavanone glucoside prunin in organic mediardquo ProcessBiochemistry vol 46 no 1 pp 94ndash100 2011

[7] M H Katsoura A C Polydera L Tsironis A D Tselepis andH Stamatis ldquoUse of ionic liquids as media for the biocatalyticpreparation of flavonoid derivatives with antioxidant potencyrdquoJournal of Biotechnology vol 123 no 4 pp 491ndash503 2006

[8] A Dıaz-Rodrıguez S Fernandez I Lavandera M Ferreroand V Gotor ldquoNovel and efficient regioselective enzymaticapproach to 31015840- 51015840- and 31015840 51015840-di-O-crotonyl 21015840-deoxynucleosidederivativesrdquo Tetrahedron Letters vol 46 no 35 pp 5835ndash58382005

[9] C H Kim M Kang H J Kim A Chatterjee and P GSchultz ldquoSite-Specific incorporation of 120576-N-crotonyllysine intohistonesrdquo Angewandte Chemie International Edition vol 51 no29 pp 7246ndash7249 2012

[10] A M Gumel M S M Annuar T Heidelberg and Y ChistildquoLipase mediated synthesis of sugar fatty acid estersrdquo ProcessBiochemistry vol 46 no 11 pp 2079ndash2090 2011

[11] L E Iglesias E S Lewkowicz R Medici P Bianchi and A MIribarren ldquoBiocatalytic approaches applied to the synthesis ofnucleoside prodrugsrdquo Biotechnology Advances vol 33 no 5 pp412ndash434 2015

[12] Z-Y Wang Y-H Bi R-L Yang et al ldquoThe halo-substituenteffect on Pseudomonas cepacia lipase-mediated regioselectiveacylation of nucleosides a comparative investigationrdquo Journalof Biotechnology vol 212 pp 153ndash158 2015

[13] Z-Y Wang Y-H Bi X-Q Li and M-H Zong ldquoInfluence ofsubstituent groups in regioselective acylation of nucleosides byNovozym 435 lipaserdquo Process Biochemistry vol 48 no 8 pp1208ndash1211 2013

[14] Y Gu and F Jerome ldquoBio-based solvents an emerging genera-tion of fluids for the design of eco-efficient processes in catalysisand organic chemistryrdquo Chemical Society Reviews vol 42 no24 pp 9550ndash9570 2013

[15] V Pace P Hoyos L Castoldi D M P Domınguez and A RAlcantara ldquo2-Methyltetrahydrofuran (2-MeTHF) a biomass-derived solvent with broad application in organic chemistryrdquoChemSusChem vol 5 no 8 pp 1369ndash1379 2012

[16] Y Simeo J V Sinisterra and A R Alcantara ldquoRegioselectiveenzymatic acylation of pharmacologically interesting nucleo-sides in 2-methyltetrahydrofuran a greener substitute for THFrdquoGreen Chemistry vol 11 no 6 pp 855ndash862 2009

[17] Z Cabrera G Fernandez-Lorente R Fernandez-Lafuente JMPalomo and J M Guisan ldquoNovozym 435 displays very differ-ent selectivity compared to lipase from Candida antarctica Badsorbed on other hydrophobic supportsrdquo Journal of MolecularCatalysis B Enzymatic vol 57 no 1ndash4 pp 171ndash176 2009

[18] R T Otto H Scheib U T Bornscheuer J Pleiss C Syldatk andR D Schmid ldquoSubstrate specificity of lipase B from Candida

antarctica in the synthesis of arylaliphatic glycolipidsrdquo Journalof Molecular Catalysis B Enzymatic vol 8 no 4-6 pp 201ndash2112000

[19] S Thusoo S Gupta R Sudan et al ldquoAntioxidant activity ofessential oil and extracts of Valeriana jatamansirootsrdquo BioMedResearch International vol 2014 Article ID 614187 4 pages2014

[20] G Paggiola A J Hunt C R McElroy J Sherwood and JH Clark ldquoBiocatalysis in bio-derived solvents an improvedapproach for medium optimisationrdquo Green Chemistry vol 16no 4 pp 2107ndash2110 2014

[21] N Doukyu and H Ogino ldquoOrganic solvent-tolerant enzymesrdquoBiochemical Engineering Journal vol 48 no 3 pp 270ndash2822010

[22] Z-G Chen D-N Zhang L Cao and Y-B Han ldquoHighly effi-cient and regioselective acylation of pharmacologically interest-ing cordycepin catalyzed by lipase in the eco-friendly solvent2-methyltetrahydrofuranrdquo Bioresource Technology vol 133 pp82ndash86 2013

[23] L Gardossi P B Poulsen A Ballesteros et al ldquoGuidelines forreporting of biocatalytic reactionsrdquoTrends in Biotechnology vol28 no 4 pp 171ndash180 2010

[24] Z-YWang N Li andM-H Zong ldquoA simple procedure for thesynthesis of potential 6-azauridine prodrugs by Thermomyceslanuginosus lipaserdquo Journal of Molecular Catalysis B Enzymaticvol 59 no 1ndash3 pp 212ndash219 2009

[25] H KWeber HWeber and R J Kazlauskas ldquorsquoWatchingrsquo lipase-catalyzed acylations using 1H NMR competing hydrolysis ofvinyl acetate in dry organic solventsrdquo Tetrahedron Asymmetryvol 10 no 14 pp 2635ndash2638 1999

[26] Z-Y Wang and M-H Zong ldquoRecognition of acyl donors bylipase CAL-B in the acylation of 6-azauridinerdquo BiotechnologyProgress vol 25 no 3 pp 784ndash791 2009

[27] X Ma R Yan S Yu Y Lu Z Li and H Lu ldquoEnzymatic acy-lation of isoorientin and isovitexin from bamboo-leaf extractswith fatty acids and antiradical activity of the acylated deriva-tivesrdquo Journal of Agricultural and Food Chemistry vol 60 no43 pp 10844ndash10849 2012

[28] J H Salem I Chevalot C Harscoat-Schiavo C Paris MFick and C Humeau ldquoBiological activities of flavonoids fromNitraria retusa (Forssk) Asch and their acylated derivativesrdquoFood Chemistry vol 124 no 2 pp 486ndash494 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


OHOHO

HO

O

OH OHHO

HO

HO

OH

OH

OH

OO

OO

Polydatin

Vinyl sorbate

5 5

49984006

998400998400

4998400998400

3998400998400

49984006

998400998400

4998400998400

39984009984002

998400998400

CALB 2-MeTHF 60∘C


1998400998400998400

2998400998400

Scheme 1 CALB-catalyzed acylation of polydatin with vinyl sorbate

Over the past several years biobased solvents that isgreen solvents derived from renewable raw materials havearoused great interest as environmentally friendly alterna-tives to traditional organic solvents in catalysis and organicchemistry particularly in biocatalytic reactions with promis-ing physicochemical properties of renewability biodegrad-ability environmental benignancy stability and so forth [14]Among these specific solvents 2-MeTHF produced fromrenewable resources of lignocellulosic biomass such as corncrops bagasse and oat hulls exerts clear advantages like thelow miscibility with water high boiling point (82∘C) neg-ligible toxicity higher stability and abiotic degradability byexposure to sunlight and air [14 15] Following the pioneeringwork of Simeo and coworkers in enzymatic acylation ofnucleosides in 2-MeTHF [16] biocatalysts have shown higheractivity and stability in this ecofriendly medium than intraditional organic solvents

Therefore in this study an effective enzymatic method in2-MeTHF is explored for preparing potential dual prodrugof sorbic acid (an unsaturated fatty acid with noticeablebactericidal and fungicidal properties) ester of polydatincatalyzed by Candida antarctica lipase B (CALB) a versatileenzyme immobilized on macroporous acrylic resin with amolecular weight of 33 kDa and approximate dimensions of10 A times 4 A times 12 A [17 18] (Scheme 1) The kinetic study of theacylation was explored in detail to better identify the effectof 2-MeTHF on the performance of the enzyme Particularlythe free radical scavenging activities of several unsaturatedacylated derivatives were also preliminarily studied

2 Materials and Methods

21 Materials CALB (Candida antarctica lipase B immobi-lized on macroporous acrylic resin 10000Ug) was fromNovozymes Co Ltd China Vinyl sorbate vinyl undeceno-ate and vinyl crotonate were obtained from TCI Poly-datin 22-diphenyl-1-picrylhydrazyl (DPPH) 2-MeTHF andt-amyl alcohol were purchased from Sigma-Aldrich All otherreagents were of analytical grade and were dried by 4 Amolecular sieves

22 Enzymatic Acylation Procedure The acylation wasconducted with polydatin (003mmol) vinyl sorbate(027mmol) anhydrous solvent (3mL) and 100mg lipasewhile stirring at 200 rpm and at a certain temperature To

determine the concentrations of the polydatin and regiose-lectivities of the acylated products aliquot fractions (50 120583L)were withdrawn at specified time intervals from the reactionsystem followed by a HPLC analysis All experiments wereconducted at least in triplicate and the errors did not exceed5 No chemical acylation was detectable as confirmed bythe control experiments

23 Operational Stability of CALB After the maximum sub-strate conversion reached during the batch acylation of poly-datin the immobilized enzyme was separated by filtrationand washed three times with 2-MeTHF or THF Then thereused prepared catalyst was added into the fresh reactionmixture (3mL) containing 003mmol polydatin 045mmolvinyl crotonate and 100mg CALB at 60∘C and 200 rpmfollowed by the assay of its activity and maximum polydatinconversion The initial activity and maximum conversionobtained in the first batchwere set to 100The residual activitywas defined as the ratio of the reused enzyme activity relativeto the original enzyme activity in the same reaction system

24 Determination of the Enzymatic Kinetic Constants Thekinetic constants of enzymatic sorboylation were investi-gated at different polydatin concentrations in 2-MeTHF (2ndash14mM) THF (5ndash50mM) acetone (4ndash16mM) and t-butanol(2ndash18mM) The reactions were carried out in 3mL solventcontaining different polydatin concentration 9 equiv of vinylsorbate and 100mg CALB at 60∘C and 200 rpm The kineticconstants (119870

119898and 119881max) were calculated from Hanes-Woolf

plots

25 Determination of the Enzymatic Apparent ActivationEnergy (119864

119886) The apparent activation energy values (119864

119886) of

enzymatic acylation in 2-MeTHF THF acetone and t-butanol were investigated Polydatin (003mmol) CALB(100mg) and vinyl sorbate (027mmol) were added intothe anhydrous solvent (3mL) and incubated at differenttemperatures (in the range of 35ndash50∘C) and 200 rpm119864

119886value

of enzymatic sorboylation was calculated according to thelinear regression analysis of the Arrhenius plot

26 NMR and HPLC Analytical Procedure Qualitative anal-ysis of the reaction mixtures was conducted by HPLC usinga 46mm times 250mm (5 120583m) Eclipse Plus-C18 column (AgilentTechnologies Industries Co Ltd USA) a UV detector at






6



21015840 + H61015840) 718ndash713 (m 1H 119869 = 180Hz H

4101584010158401015840)


2101584010158401015840) 687 (t 1H Hvinyl-2) 681

(d 1H 119869 = 56Hz H31015840) 670 (br s 1H H

51015840) 663 (br s 1H

H2) 636 (t 1H 119869 = 16Hz H

6) 616ndash612 (m 1H H

5101584010158401015840)

580 (dd 1H 119869 = 12 12Hz H4) 542 (br s 1H OH

210158401015840)

536 (br s 1H OH310158401015840) 524 (br s 1H OH

410158401015840) 494 (d 1H

119869 = 52Hz H110158401015840) 447 (d 1H 119869 = 72 H

610158401015840-1) 411 (dd

1H 119869 = 52 52Hz H610158401015840-2) 374ndash371 (m 1H H

510158401015840) 339ndash

336 (m 1H H210158401015840) 332ndash330 (m 1H H

310158401015840) 325ndash322 (m 1H

H410158401015840) 171 (d 3H 119869 = 44Hz H

6101584010158401015840) 13C NMR (DMSO-119889

6)

120575 16677 (C1101584010158401015840) 15905 (C

3) 15886 (C

5) 15785 (C

41015840) 14564

(C3101584010158401015840) 14043 (C

4101584010158401015840) 13982 (C

1) 12986 (C

21015840 + C61015840) 12895

(C11015840) 12840 (Cvinyl-1) 12576 (Cvinyl-2) 11875 (C

2101584010158401015840) 11605

(C31015840 +C51015840) 10762 (C

6) 10479 (C

2) 10337 (C

110158401015840) 10055 (C

4)

7689 (C210158401015840) 7429 (C

510158401015840) 7362 (C

310158401015840) 7059 (C

410158401015840) 6417 (C

610158401015840)

6024 (C5101584010158401015840) 1877 (C

6101584010158401015840)







2

3

4

5

6In

itial

reac

tion

rate

(mM

h)


20

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)


(a)


40 45 50 55 60 65 7035Temperature (∘C)

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)

2

4

6

8

Initi

al re

actio

n ra

te (m

Mh

)

(b)


20

40

60

80

100

Max

imum

conv

(

)


20

40

60

80

100

Regi

osele

ctiv

ity (

)

(c)






2-MeTHFTHF

0

20

40

60

80

100

120Re

lativ

e act

ivity

()

2 3 4 5 6 7 8 9 10 11 121Batch

(a)

0

20

40

60

80

100

120

Relat

ive m

axim

um co

nv (

)

2-MeTHFTHF

2 3 4 5 6 7 8 9 10 11 121Batch

(b)

















Solvent

549

527

507


Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)



4 Conclusions


119898and 119864



60

70

80

90

100

Scav

engi

ng ac

tivity

()


Polydatin






Competing Interests




Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






6



21015840 + H61015840) 718ndash713 (m 1H 119869 = 180Hz H

4101584010158401015840)


2101584010158401015840) 687 (t 1H Hvinyl-2) 681

(d 1H 119869 = 56Hz H31015840) 670 (br s 1H H

51015840) 663 (br s 1H

H2) 636 (t 1H 119869 = 16Hz H

6) 616ndash612 (m 1H H

5101584010158401015840)

580 (dd 1H 119869 = 12 12Hz H4) 542 (br s 1H OH

210158401015840)

536 (br s 1H OH310158401015840) 524 (br s 1H OH

410158401015840) 494 (d 1H

119869 = 52Hz H110158401015840) 447 (d 1H 119869 = 72 H

610158401015840-1) 411 (dd

1H 119869 = 52 52Hz H610158401015840-2) 374ndash371 (m 1H H

510158401015840) 339ndash

336 (m 1H H210158401015840) 332ndash330 (m 1H H

310158401015840) 325ndash322 (m 1H

H410158401015840) 171 (d 3H 119869 = 44Hz H

6101584010158401015840) 13C NMR (DMSO-119889

6)

120575 16677 (C1101584010158401015840) 15905 (C

3) 15886 (C

5) 15785 (C

41015840) 14564

(C3101584010158401015840) 14043 (C

4101584010158401015840) 13982 (C

1) 12986 (C

21015840 + C61015840) 12895

(C11015840) 12840 (Cvinyl-1) 12576 (Cvinyl-2) 11875 (C

2101584010158401015840) 11605

(C31015840 +C51015840) 10762 (C

6) 10479 (C

2) 10337 (C

110158401015840) 10055 (C

4)

7689 (C210158401015840) 7429 (C

510158401015840) 7362 (C

310158401015840) 7059 (C

410158401015840) 6417 (C

610158401015840)

6024 (C5101584010158401015840) 1877 (C

6101584010158401015840)







2

3

4

5

6In

itial

reac

tion

rate

(mM

h)


20

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)


(a)


40 45 50 55 60 65 7035Temperature (∘C)

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)

2

4

6

8

Initi

al re

actio

n ra

te (m

Mh

)

(b)


20

40

60

80

100

Max

imum

conv

(

)


20

40

60

80

100

Regi

osele

ctiv

ity (

)

(c)






2-MeTHFTHF

0

20

40

60

80

100

120Re

lativ

e act

ivity

()

2 3 4 5 6 7 8 9 10 11 121Batch

(a)

0

20

40

60

80

100

120

Relat

ive m

axim

um co

nv (

)

2-MeTHFTHF

2 3 4 5 6 7 8 9 10 11 121Batch

(b)

















Solvent

549

527

507


Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)



4 Conclusions


119898and 119864



60

70

80

90

100

Scav

engi

ng ac

tivity

()


Polydatin






Competing Interests




Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2

3

4

5

6In

itial

reac

tion

rate

(mM

h)


20

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)


(a)


40 45 50 55 60 65 7035Temperature (∘C)

40

60

80

100

Con

v (

) or r

egio

sele

ctiv

ity (

)

2

4

6

8

Initi

al re

actio

n ra

te (m

Mh

)

(b)


20

40

60

80

100

Max

imum

conv

(

)


20

40

60

80

100

Regi

osele

ctiv

ity (

)

(c)






2-MeTHFTHF

0

20

40

60

80

100

120Re

lativ

e act

ivity

()

2 3 4 5 6 7 8 9 10 11 121Batch

(a)

0

20

40

60

80

100

120

Relat

ive m

axim

um co

nv (

)

2-MeTHFTHF

2 3 4 5 6 7 8 9 10 11 121Batch

(b)

















Solvent

549

527

507


Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)



4 Conclusions


119898and 119864



60

70

80

90

100

Scav

engi

ng ac

tivity

()


Polydatin






Competing Interests




Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2-MeTHFTHF

0

20

40

60

80

100

120Re

lativ

e act

ivity

()

2 3 4 5 6 7 8 9 10 11 121Batch

(a)

0

20

40

60

80

100

120

Relat

ive m

axim

um co

nv (

)

2-MeTHFTHF

2 3 4 5 6 7 8 9 10 11 121Batch

(b)

















Solvent

549

527

507


Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)



4 Conclusions


119898and 119864



60

70

80

90

100

Scav

engi

ng ac

tivity

()


Polydatin






Competing Interests




Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Solvent

549

527

507


Ea (KJmol)

305 310 315 320300minus12

minus08

minus04

00

04

08

LgV0

1T (lowast10minus3K

minus1)



4 Conclusions


119898and 119864



60

70

80

90

100

Scav

engi

ng ac

tivity

()


Polydatin






Competing Interests




Acknowledgments


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article enzymatic synthesis of sorboyl-polydatin...

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