Synthesis of biosafe isosorbide dicaprylate ester plasticizer by lipase in a solvent-free system and its sub-chronic toxicity in mice

Article abstract of DOI:10.1039/c5ra27537g

In this study, biosafe isosorbide dicaprylate ester based plasticizer was prepared using renewable feedstock with lipase in a solvent-free system. Different kinds of water removal methods and some important factors including molar ratio of the substrates, reaction temperature and catalyst loading were investigated. Bubbling dried air was determined to be the most effective water removal method. The activation energy (E) of hydrolysis and esterification to form di-substituted isosorbide were examined to determine the limiting step in the synthesis of isosorbide dicaprylate ester. The E_hydrolysis (25.51 kJ mol^-1) was found to be higher than E_synthesis (35.65 kJ mol^-1), demonstrating that the formation of diester from monoester is the critical step in the process. The lipase can be recycled up to 16 times while 80% diester was maintained. Additionally, the properties of poly(vinyl chloride) PVC blends plasticized with isosorbide ester as secondary plasticizer was studied. The results indicated that the thermal stability of plasticized PVC blends was improved, and the tensile strength was increased. Furthermore, the sub-chronic toxicity study in mice showed that the isosorbide ester was safe, indicating its great potential in industrial applications as a plasticizer.

Full text of DOI:10.1039/c5ra27537g

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Synthesis of the biosafety isosorbide dicaprylate ester
plasticizer by lipase in solvent-free system and its
sub-chronic toxicity in mice
Citethis: DOI: 10.1039/ x0xx00000x
Caixia Cuia, Yueju Zhenb, Junge Quc, Biqiang Chen* a and Tianwei Tan* a
Received 00th January 2012,
Accepted 00th January 2012
In this study, biosafety isosorbide dicaprylate ester based plasticizer was prepared using
renewable feedstock with lipase in a solvent-free system. Different kinds of water removal
methods and some important factors including molar ratio of the substrates, reaction
temperature and catalyst loading were investigated. Bubbling dried air was determined to be
the most effective water removal method. The activation energy (E) of hydrolysis and
esterification to form di-substituted isosorbide were examined to determine the limiting step in
the synthesis of isosorbide dicaprylate ester. The Ehydrolysis (25.51 KJ/mol) was found to be
higher than Esynthesis (35.65 KJ/mol), demonstrating that the formation of diester from
monoester is the critical step in the process. The lipase can be recycled up to 16 times while
80% diester was maintained. Additionally, the property of poly (vinyl chloride) PVC blends
plasticized with isosorbide ester as secondary plasticizer was studied. The results indicated that
the thermal stability of plasticized PVC blends was improved, and the tensile strength was
increased. Furthermore, the sub-chronic toxicity study in mice showed that the isosorbide ester
was safe, indicating its great potential in industrial application as plasticizer.
DOI: 10.1039/ x0xx00000x
www.rsc.org/
he atlh when ti is use d ni food pack ni g, aut omot ive
product s, ni se ct er pe ellnt s, b lood st orage b ags an d
I. Introduction
O ve r the
past
de cade,
product s wh ich
aer
me dical de vice s 8,
be came impe rat ive.
.
Th us the b iob- ase d plast iczie r
9
en viron ment ally an db iologically frein dlyh ave att racte d
ni cer ase d attent ion 1 -3 Source s of raw mate rials an d
.
The de ve lopment ofb iob- ase d, e spe cially ve get ab el an d
ce er alb- ase d, che micals h as the potent ial t o er duce
pet roel um con sumpt ion ni the che micalni dust ry an d also
t o open new h igh -value -adde d makret s1 0. I sosobr ide
product ion proce sse s aer impotr ant t othe ove rallq uatli y
of such product s. Biomass st ock s aer ab un dant cabr on -
ne ut ral ernew ab el er source s wh ich can be gene rate d
dier ct ly an d rapidly from solarene rgy. Spe cifically, the
use ofb iologically de rive d raw mate rials an db io-cat alyst s
is amon gthe most promisni g approache s for de ve lopni g
b io-frein dly product s 5. The comperhen sive ut izli at ion of
b iob- ase d mate rials an dthe de ve lopment ofb io-er fnie ry
proce sse s could mni imzie w aste
en viron ment wh eil simutl ane ously ni cer asni ge con omic
1,4: 3, 6-dianh ydro-D-glucti ol
is an impotr ant b io-
efe dst ock asti is ch ira,l aswe ll asn on t- oxic . The n on -
11
t oxic ch aracte r of isosobr ide eln ds ti t o applicat ion s ni
4,
pack agni g, cosmet ics an d me dicnie
.
De sptie ti s
favorab el prope trei s, isosobr ide h as been ut izlie d ni
er lat ive ly efw applicat ion s. C urernt ly, ti s large st
1 2
an d prote ct the
comme rcial use isni the product ion of isosobr ide n ti rate
1 3, wh ich is use dt ot er at an gni a. As ti isb iode gradab el
an dthe rmally st ab el, isosobr ide h as alsobeen appeli d as
a polye ste rb ackb one use dni the synthe sis ofthe rmoset s
bene fti6, 7
Poly
.
vni yl ch loride
PVC
isthe se con d large st
man ufact uer s er sni ni the w orld, an d ti s product s aer
w ide ly use dni pack agni g, t oys, floorni g, cards an db ags.
Plast iczie r plays an impotr ant roel ni improvni g the
fel xbi itli y an d proce ss ab itli y of PVC. Phth alate s isthe
mani plast iczie r addti ive s of PVC, wh ich h asbeen use d
aroun dthe w orld for man y ye arsbe cause ofti se xce ellnt
plast iczi ni ge fef ct, b ut phth alate s may migrate from PVC
product st oen viron ment when the mate rials cont act w tih
b iological fluids, wh ich w illt ake potent ial risk st oh uman
.
Kn own synthe se s of isosobr ide -de rive d product s aer
14
otfen de pen dent on act ivate d di-acid de rivat ive s orLew is
acid cat alyst s coupel dw tih the er moval of low mass
con den sat ion product s . The se proce sse s produce large
1 5
q uant tiei s ofw aste w ate r an dthe h armful cat alyst s an d
b y-product s aer difficutl t o er move from the de sier d
product s.
Th us, en viron ment -frein dly b io-er fnie ry
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

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proce sse s aer the most promisni g product ion proce sse s enz ymat ic solvent -fere con dti ion s usni g Novo4 35 asthe
t o acce sst o isosobr ide -de rive d che micals. cat alyst. The e xpe riment route is illust rate dni sche me 1.
Journal Name
DOI: 10.1039/C5RA27537G
L ipase cat alyze d e ste rificat ion, h ydrolysis, alcoh olysis Durni gthe e ste rificat ion, se ve ral fact orswe er foun dt o
an d acidolysis er act ion s aer amon gthe most promisni g h ave sign ificant ni fluen ce ove r the er act ion content.
an d en viron ment ally frein dly atle nr at ive s t o t radti ion al The se fact ors aer the meth od for er movni gw ate ,r the
che mical meth ods . C ompaer dt o che mical cat alyst s, st oich iomet ry of caprylic acid an d isosobr ide, the er act ion
1 6
lipase s er act un de r milde r con dti ion s, h ave b roade r te mpe rat uer, an d the enz yme loadni g. The act ivat ion
sub st rate spe cificti y, er duce un de sirab el b y-product s an d ene rgy forthe h ydrolysis an de ste rificat ion of isosobr ide
erq uier lowe r ene rgy ni put
.
C an dida ant arct ica dicaprylate e ste rw as dete rmnie dt oe xplani the limtini g
L ipase B C AL B ni patr icularh asbeen foun dt obe fact or ni ach ei vni gh igh e ste r content an dthe er foer t o
ve rsat eil b iocat alyst ni the food, pape ,r ph armace ut ical opt imzie the er act ion wh eil er ducni g cost s. The
an d cosmet ic ni dust rei s an d C AL B-cat alyze d er cyclab itli y an d ope rat ion al st ab itli y of Novo4 35 un de r
e ste rificat ion h as er cent ly grown ni popularti y20-22 The the solvent -fere con dti ion swe er also ni ve st igate d. The
prope trei s of poly vni yl ch loride PVC b eln ds
plast iczie dw tih isosobr ide e ste r as se con dary plast iczie r
st udei d. F ni ally, the sub -ch ron ic t oxicti y of
17, 1 8
a
,
1 9
.
er sutl ant b iob- ase d e ste rs aer e co-frein dly, sni ce the
compoun ds aer gene rate d from ernew ab el er source s, the
synthe sis produce s mni imal w aste,
an d the y aer we er
b iode gradab el.
isosobr ide e ste rw aste ste dni mice.
Few pape rsh ave er ef rer dt othe isosobr ide dei ste r as
plast iczie r an dthe enz ymat ice ste rificat ion of isosobr ide
w tih aliph at ic acids: D. Muke sh et a.l er potre d lipoz yme
I M-20-cat alyze de ste rificat ion of isosobr ide oel ate ni the
II. Materials and Methods
A. Mate rials an d Eq uipment
per sen ce of silica ge lt o er move w ate r . C. Ce cutt iet a.l
er potre d the er act ion of isosobr ide w tih fatt y acyl
ch loride st o yei ldthe dei ste rw tih out cat alyst sni pyridnie
23
The cat alyst Novoz ym4 35w as purch ase d from Novo
Nordisk A/S
Den makr . I sosobr ide
purti y: 98%
w as suppeli db y Sni oph armChe micalRe agent C o., Lt d.
an d DMF
.
Moer er cent ly, El Boulifiet a.l er potre d
24
C aprylic acid
99% w as from Be inji g Che mical
enz ymat ic product ion ofthe mon oe ste r from isosobr ide
F act ory, Be inji g, Ch ni a. Allthe er agent s an d solvent s
use d ni the e xpe riment swe er of an alyt ical grade. The
e ste rificat ion an dh ydrolysis er act ion swe er carrei d out ni
an d ricni oel ic acid . In the se st udei s, the er st ricte dw ate r
25
adsobr ni g capacti y of silica ge l an dthe solvent
pyridnie
an d DMF limti s the scalab itli y of th is meth od t o
paraell .l Polyvni yl ch loride
PVC
w as obt anie d b y
ni dust rial applicat ion s, an dthe fatt y acyl ch loride er agent
isn ot a de sier d sub st rate forthe synthe sis ofe ste rs
be cause the ope rat ion proce ss isn ot saef.
Sh an don g qji ni gChe micalC o., Lt d. Kun mni g mice we er
purch ase d from Expe riment al An imalCente r ofCh nie se
Drug an d Biological Product s.
B. Enz ymat ic synthe sis of isosobr ide dei tse r
Expe riment swe er carrei d out ni a1 00 mltw on-e ck flask.
The suti ab el e ste rificat ion er act ion s of isosobr ride an d
caprylic acidw tih Novo4 35 lipase we er e xamnie d un de r
difef ernt w ate rer moval con dti ion s the er act ion e xpose d
t o ai,r the er act ion un de r vacuum, an d the er act ion
b ubb el dw tih drei d air , caprylic acidt o isosobr ide molar
O
H
O
OH
O
O
OH
H
H
O
O
O
O
O
H
HO
rat ios ni the solvent -fere syste m
er act ion te mpe rat uer s
lipase loadni gs
2, 2. 5, 3, an d4
,
,
50, 60, 7 0, an d7 8 C
an d
3. 5, 5, 6. 5, an d 8 % w w/
. F or
h ydrolysise xpe riment s, the e ste rificat ion product swe er
er acte d w tih w ate r un de r the opt imzie d e ste rificat ion
con dti ion s 29. 3 g dei ste ,r 3 gw ate ,r 1. 97 g Novoz ym
4 35, 300 rpm for1 05 mni at 60 C
. When dete rmni ni g
the act ivat ion ene rgy ofthe e ste rificat ion an dh ydrolysis
er act ion s, the er act ion swe er con ducte d cont ni uously ni
the er act or for 60 mni. The er act ion con dti ion ofthe
e ste rificat ion w as as follow s: caprylic acidt o isosobr ide
Sche me 1. The e xpe riment route w ay
To ove rcome the limti at ion s nit roduce db ythe use of
solvent s an d silica ge l an d t o bette r un de rst an d the
crti ical fact or for synthe szi ni g isosobr ide dei ste ,r we
per sent an e co-frein dly synthe sis of isosobr ide dei ste .r
I sosobr ide an d caprylic acid we er e ste rifei d un de r
molar rat io1: 2. 5, 8% w w/
lipase loadni g amount, 300
rpmw tih b ubb nli g drei d air at 4 0, 50 or 60 C. An dthe
er act ion con dti ion of the h ydrolysis er act ion s w as as
follow s: dei ste rt ow ate r molar rat io1: 2. 5, 8% w w/
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA27537G
lipase loadni g amount, 300 rpmw tih b ubb nli g drei d air at The mice ni cont rol group on ly er ce ive d veh icel
0. 5%
30, 4 3 or 50 C. The w ate r act ivti y an dthe te mpe rat uer C MC -Nani dist iell dw ate r , whe er as othe r groupswe er
ofthe air at the ni elt an d out elt we er me asuer d ni the t er ate d dailyw tih dei ste r at 5, 1 0 or 20 μl/g mice /day for
gase ous ph ase b y HygroC lip 2 sen sors R ot ron ic, 28 days, er spe ct ive ly. Atfe r 28 days, the b lood rout nie,
Sw tize rlan d
an d mon ti oer d on
a
compute .r The
proth romb ni t ime
PT ,
act ivate d patr ial clott ni g
TT ,
mon oe ste r an d dei ste r content swe er an alyze db y gas enz yme live t ime
APTT , th romb ni t ime
ch romat ograph y GC
C. GC An alysis
.
fbi rni ogen FI B an db loodb ioche mical AL T an d BU N
we er dete rmnie d. Mice live r an d mael mice te st iswe er
ob se rve db y opt ical microscope. Allthe e xpe riment swe er
Aqli uot swe er t aken fromthe er act ion s at sche duel dt ime s pe rforme d ni complian ce w tih the er el vant law s an d
an d imme diate lyhe ate dt oen suer enz yme de act ivat ion. ni st ti ut ion al guide nlie s, the ni st ti ut ion al commtitee h as
The el ve ls of fatt y acid, mon o,- an d di-sub st ti ute de ste rs approve dthe e xpe riment s, an dthe ni forme d con sent w as
ni the er act ion mixt uer we er q uant ifei d usni g a Trace
1 300 gas ch romat ograph The rmo sceint ific, U SA
eq uippe dw tih a DB1-ht capillary column 30 m × 0. 25 Tab el 1. The proej ct ofthe isosobr ide dei ste rt oxicti yni
obt anie d fore xpe riment at ion w tih h uman sub ej ct s.
mm × 0.1 μm; J& W Sceint ific, U SA an d a flame ion zi ni g mice
dete ct or FI D . The column te mpe rat uer w ashe ld at
1 20 C an dthen rampe dt o1 80 C at 30 C /mni an dt o
320 C at 4 5 C /mni whe er ti w ashe ld for 2 mni. The
te mpe rat uer s ofthe ni ej ct or an d dete ct orwe er b oth set at
320 C. The ertent ion t ime of caprylic acid, isosobr ide,
an d mon oe ste r an d dei ste rw as1. 387 mni, 1. 533 mni,
3. 560 mni an d 5. 308 mni, er spe ct ive ly. The molar
fract ion s of fatt y acid, isosobr ide, mon oe ste r an d dei ste r
High dose
μg/g
20
Middel dose
1 0 μg/g
L ow dose
μg/g
5
mice /day
mice /day
mice /day
C ont rol
3ef mael
mice s
we er calculate d.
3ef mael mice s
3 mael mice s
3ef mael mice s
3 mael mice s
0. 5%C MC -
Nani dist iell d
w ate r
D. Per parat ion ofb eln ds an d me asuer ment s
PVC b eln dswe er me tle d at 1 65 C for 5 mni at 50 rpm
usni g a PolyL ab Toqr ue hre omete r Hakke In st rument
C rop., Ge rman y . PVC er sni w as per t-er ate db y dryni g
at 60 C for 2h t oe limni ate possbi el ab sobre dw ate r on
the surface of patr icel. F ormulat ion s use d for per parni g
plast iczie d PVC b eln ds we er 7 0 g PVC an d 30 g
isosobr ide dei ste .r Dumbbe ll-sh ape d sampel s ofb eln ds
we er molde d on a Mni iJetII Micro-ni ej ct ion moldni g
3 mael mice s
3ef mael
mice s
3ef mael mice s
3 mael mice s
3ef mael mice s
3 mael mice s
Sampel
mach nie
t o GB/T17 037.1 1- 997
Hakke In st rument C rop. Ge rman y accordni g
Ch ni a . Moun dni g con dti ion s
3 mael mice s
we er set at 1 65 C for 5 mni at 550b a.r
TGAw as carrei d out ni a TG209F1 TGAthe rmal an alysis
ni st rument s Netz sch In st rument C rop., Ge rman
at mosphe er 50 ml/mni at ahe at ni g rate of1 0 C /mni.
The sampel swe er put nit o plat ni um pan s an d scanne d
from amb eint te mpe rat uer t o 600 C.
ni N
2 Results and Discussions
A. Efef ct ofthe w ate rer moval meth ods
Ten seil modulus, ten seil st ern gth, an de lon gat ion at b er ak
A
we er
Ch ni a
Un ive rsal Te st ni g Mach nie
dete rmnie d accordni g t o GB/T 1 04 0.1 -2006
un de r amb eint con dti ion s, usni g E4 3.1 04
MTS In st rument C rop.,
Ch ni a . The er potre d value swe er the ave rage of at el ast
th ere sampel s. Hardne ssw as dete rmnie d usni g a HT-
651 0D Sh oer duromete r
Sh an gh ai Jei lun, Ch ni a
accordni gt o GB/T 3398. 2- 2008 Ch ni a .
E. Sub -ch ron ict oxicti y st udy
The mice b oth mael an d ef mael we er we ighe d upon
arriva,l an dthen ran domly dist rbi ute d nit o four groups.
The dei ste rw as admni iste er dt othe mice on ce a day.
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DOI: 10.1039/C5RA27537G
w tih out drei d airb ubb el d nit othe syste m. Howe ve ,r as
the er act ion t ime ni cer asni g, the w ate r act ivti y cont ni ue d
t oni cer ase t othe eq uibli rium 4 2% ab out 2h w tih out
drei d airb ubb nli g nit othe syste m, an d ti de cer ase dt o
1 2% an dke pt st ab el when the drei d airw asb ubb el dnit o
the syste m. It prove dth at the drei d air could er move the
produce dw ate re fficeint ly, an d er gulate the e ste rificat ion
ni solvent -fere syste m. The er foer, the b ubb el d er act or is
the opt imal one. Atfe r 24 h, the er act ion er ache d
eq uibli rium so a er act ion t ime of 24 h w as use d t o
Without control
Exposed to air
Under vacuum
90
80
70
60
50
40
30
20
10
0
Bubbled with dried air
.
opt imzie er act ion con dti ion s an denz yme loadni g
B. Efef ct ofer act ion con dti ion s on dei ste r content
0
5
10 15 20 25 30 35 40 45 50
Time (h)
1
Efef ct of sub st rate s molar rat io
The e fef ct of ch an gni g the rat io of caprylic acid t o
isosobr ide ove rthe ran ge of 2:1 t o4:1 on dei ste r content
w asni ve st igate d. F ig. 2A sh ow sth at the h ighe st content
of dei ste rw as ob se rve d usni g a molar rat io caprylic
B
Bubbled with dried air
Exposed to air
50
40
30
20
10
0
acidt o isosobr ide
of 2:1. When the molar rat iow as
h ighe rth an 2:1, w tih the ni cer ase ofthe molar rat io, the
format ion of dei ste r de cer ase d, possbi ly due t o the
enz yme nih bi ti ion b y e xce ss fatt y acid. The dei ste r
content w as74 %, 64 %, 56%, an d4 8%when the molar
rat io w as 1: 2, 1: 2. 5, 1: 3, an d 1:4. The amount of
isosobr ide use dw ashe ld con st ant ni the se er act ion s,
wh eil the caprylic acid amount varei dw tih variat ion s ni
sub st rate molar rat io. The er foer, w tih a fixe d molar rat io,
the er lat ive lyh ighe r isosobr ide con cent rat ion el dt o a
ger ate r con ve rsion of isosobr ide t oe ste .r The opt imal
molar rat iow as dete rmnie dt obe 2:1 forthe format ion of
isosobr ide dei ste .r
0
5
10
Time (h)
15
20
25
F ig. 1
A
Efef ct s ofthe w ate r er moval meth ods on
The w ate r content ofthe er act ion
dei ste r content.
B
e xpose dt o air an dthe er act ion b ubb el dw tih drei d ai.r
Re act ion con dti ion s: 9. 855 g isosobr ide 24. 3 g caprylic
acid, 1. 97 g Novoz ym4 35, at 300 rpm for 50h at 50 C.
2
Efef ct ofte mpe rat uer
In enz yme cat alyze d er act ion s, te mpe rat uer plays an
impotr ant roel. Re act ion te mpe rat uer afef ct sthe solub itli y
ofthe sub st rate s an d product s, masst ran sef ,r er act ion
rate s, er act ion eq uibli ria, aswe ll asthe st ab itli y ofthe
enz yme. As sh own ni F ig. 2B, ni cer asni gthe er act ion
te mpe rat uer ab ove 50 C, er sutle d ni a corer spon dni g
ni cer ase ni the rate of e ste rificat ion an d ni product
content w tih the maximum content ob se rve d at 60 C
80% . In cer asni g the te mpe rat uer be yon d 60 C
er sutle dni a tlit el de cer ase ni product content, an dwhen
the er act ion te mpe rat uer er ache d 7 0 C, the dei ste r
In organ ic syste ms, the h ydrat ion of lipase isne ce ssary
for cat alyt ic fun ct ion. Howe ve ,r w ate r is ab y-product of
e ste rificat ion er act ion s an dthe er act ion rate an de ste r
content w ill de cer ase if w ate r is accumulate d ni the
er act ion me dium. It isth usne ce ssaryt o er move w ate r
fromthe e ste rificat ion t o obt ani h igh er act ion rate s an d
e ste r content. As sh own ni F ig. 1 A , difef ernt w ate r
er moval meth odswe er use d, an dthe y ni cer ase dthe
e fficein cy of dei ste r synthe sis as compaer d t o the
er act ion w tih out w ate r er mova.l Spe cifically, the dei ste r
content h ad a tlit el de cer ase
opt imaler act ion te mpe rat uer w as 60 C 27
1.7 % . The er foer the
.
content
64 % ish ighe rwhen the er act or isb ubb el d
w tih airth an when ti ise xpose dt o air 23% an dhe ld
un de r vacuum 21 % atfe r 24 h, an dti is much h ighe r
th an w tih out er moval of w ate r
6% . F ig. 1
B
sh owe dthe t ime -course profeil s ofthe w ate r act ivti y ni
difef ernt er act ion syste ms. It is ob viousth at the w ate r
act ivti yw as ni cer ase d ni the first 30 mni ute sw tih or
4 | J. Name., 2012, 00, 1-3
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Re act ion con dti ion s: 9. 855 g isosobr ide, 1 9.44 g, 24. 3 g,
29.1 6 g, 38. 88 g caprylic acid, 1. 97 g Novoz ym4 35, at
1:2
1:2.5
1:3
80
60
40
20
0
300 rpm for 24 h at 50 C.
B
Efef ct ofe ste rificat ion
1:4
te mpe rat uer on dei ste r content. Re act ion con dti ion s:
9. 855 g isosobr ide, 1 9.44 g caprylic acid, 1. 97 g Novoz ym
4 35, at 300 rpm for 24 h un de r 50, 60, or7 0 C.
C
A
Efef ct ofenz yme loadni g on dei ste r content. Re act ion
con dti ion s: 9. 855 g isosobr ide, 1 9.44 g caprylic acid, at
300 rpm for 24 h at 60 C w tih 3. 5%, 5%, 6. 5% an d 8%
0
0
0
5
10
Time (h)
15
15
15
20
20
20
25
w w/
Novoz ym4 35.
D
The content of isosobr ide,
caprylic acid, mon oe ste r an d dei ste rni the e ste rificat ion
proce ss. Re act ion con dti ion s: 9. 855 g isosobr ide, 1 9.44 g
50
60
70
80
60
40
20
0
caprylic acid, at 300 rpm for 24 h at 60 C, w tih 1. 97
Novoz ym4 35.
g
3
Efef ct ofenz yme loadni g
B
In cer ase d cat alyst loadni g alsoh ad a posti ive ni fluen ce
on e ste r content ove rthe e xpe riment al ran ge, whe er the
enz yme loadni gw asni cer ase d from 3. 5%t o 8% w w/
.
5
10
Time (h)
25
The e fef ct ofni cer asni g lipase loadni g is illust rate dni F ig.
2C. The content of dei ste r an dthe er act ion rate we er
maximzie dwhen the lipase con cent rat ion
8% w asthe
h ighe st. Un de rthe se con dti ion s, the er act ion er ache d
eq uibli riumq uick ly, w tih a dei ste r content of 80%. Th is
illust rate sth at h igh cat alyst loadni gs aer ne ce ssary for
the synthe sis of a dipolar e ste r w tih h ydroph ilic an d
3.5 %
5 %
6.5 %
8 %
80
60
40
20
0
h ydroph ob ic compoun ds,
as h as been
er potre d
per viously . As sh own ni F ig. 2D, the be llt- ype curve of
28
mon oe ste r me an sth at the dei ste r is gene rate d ste pw ise.
Be foer 1. 25h, due t othe h igh isosobr ide con cent rat ion,
the syste m cont ani s a large amount of mon oe ste r
28% . Howe ve ,r on ly a small amount of isosobr ide
C
5
10
Time (h)
25
caprylate dei ste r forme d ni th ist ime
9% . As the
con cent rat ion of mon oe ste rni cer ase s, the product ion rate
of dei ste r alsoni cer ase s an d utl imate ly er ache s an 81 %
content.
80
60
40
20
0
caprylic acid
Isosorbide
monoester
diester
C. Dete rmni at ion of act ivat ion ene rgy
The act ivat ion ene rgy for the h ydrolysis an d the
e ste rificat ion
of di-sub st ti ute d isosobr ide e ste rs b y
D
Novoz ym 4 35 we er st udei d un de r opt imzie d er act ion
con dti ion s ove r ate mpe rat uer ran ge of4 0-60 C usni g
the Arhren ius eq uat ion as given ni Eq.
1 , wh ich
er per sent sthe te mpe rat uer de pen den ce ofer act ion rate:
1
E
0
5
10
15
20
25
k w=hAe0er×ek−
RTis the er act ion rate con st ant,
A
is a per -
Time (h)
0
e xponent ial fact o,r E isthe act ivat ion ene rgy KJ/mol ,
F ig.
A
2.
Efef ct of er act ion con dti ion s on dei ste r content.
Efef ct of sub st rate s molar rat io on dei ste r content.
R
isthe gas con st ant
8. 314 J/mol/K , an d T isthe
act ivat ion ene rgy for the
te mpe rat uer K .
The
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h ydrolysis ofthe dei ste rw as calculate d usni g er act ion
te mpe rat uer s of 30, 4 3 an d 50 C an d 29. 3 g dei ste ,r 3 g
w ate r an d1. 97 g Novoz ym4 35 mon ti oer d from 0t o 60
mni. The act ivat ion ene rgy forthe synthe sis of dei ste r
w as calculate d usni g er act ion te mpe rat uer s of4 0, 50 an d
60 C an d1 9.44 g caprylic acid, 9. 85 g isosobr ide an d
1. 97 g Novoz ym4 35 mon ti oer d from 0t o1 20 mni. In
caprylic acid
isosorbide
monoester
diester
100
80
60
40
20
0
e ach case, the dei ste r content
GC.
% w as calculate db y
The dei ste r content w as plotte dw tih er spe ct t ot ime t o
e st imate the rate con st ant
for h ydrolysis an d for the
k
at difef ernt te mpe rat uer s
synthe sis of dei ste ,r
er spe ct ive ly. The nlie ar Arhren ius plot w as use d t o
calculate the act ivat ion ene rgy forb oth the
Tab el 2. R ate con st ant forh ydrolysis an d synthe sis
of dei ste rb y Novoz ym4 35 at difef ernt te mpe rat uer s
E
0
20
40
60
80
100
120
k
Time (min)
F ig. 3. Efef ct of h ydrolysis er act ion on caprylic acid,
mon oe ste ,r isosobr ide, an d dei ste r content. Re act ion
con dti ion s: 29. 3 g dei ste ,r 3 gw ate ,r 1. 97 g Novoz ym
4 35, at 300 rpm for1 05 mni at 60 C.
k
Ea
KJ/mol
T
K
mmohl/
nl k
As sh own ni F ig. 3, ni the proce ss of isosobr ide dei ste r
h ydrolyz at ion, the content of dei ste r de cer ase dq uick ly
w tih er act ion t ime, the content of caprylic acid an d
mon oe ste r alsoni cer ase dq uick ly. Howe ve ,r the er act ion
er ache deq uibli rium 50 mni late rwhen the content ofthe
caprylic acid, mon oe ste ,r an d dei ste rbe came ab out 30%,
51 %, an d1 8%. The er foer the mon oe ste r is st ab el an d
doe sn ot un de rgoh ydrolysist o caprylic an d isosobr ide
e asily. The content s ni the h ydrolysis syste m an dthe
act ivat ion ene rgy ofthe h ydrolysis er act ion de mon st rate
th at the dei ste r can h ydrolyze t othe mon oe ste r an d
caprylic acide asily, whe er asthe synthe sis of dei ste r is
moer difficutl. Togethe ,r the se er sutl s de mon st rate th at
303
31 6
323
31 3
323
333
1 0. 91
17. 29
20.1 9
1. 01
2. 39
2. 85
3. 01
Hydrolysis
er act ion
25. 51
0. 007 5
0. 55
0. 83
Etse rificat io
n
1.74
35. 65
2. 29
h ydrolysis an d synthe sis of isosobr ide dei ste rw tih lipase
accordni gt o Eq. usni gthe value s given ni Tab el 2.
Act ivat ion ene rgy of 25. 51 KJ/mol an d 35. 65
KJ/molwe er calculate d forthe h ydrolysis an d synthe sis of
di-sub st ti ute d isosobr ide, er spe ct ive ly. The for
h ydrolysis of the dei ste r w as h ighe r th an th at for
synthe szi ni g dei ste ,r an dthe er w asn oh ydrolysiser act ion
w tih out enz yme. Th is ni dicate s th at the format ion of
dei ste r erq uier s moer ene rgyth an the dei ste rh ydrolysis
synthe szi ni g isosobr ide
dicaprylate e ste r from the
1
corer spon dni g mon oe ste r is the crti ical ste p ni the
synthe sis.
E
E
D. Re usab itli y ofenz yme
The ope rat ion al st ab itli y ofthe lipase is ve ry impotr ant for
the pract icab itli y ofthe synthe sis of isosobr ide dei ste rni
ni dust rial applicat ion s. The er usab itli y ofthe lipase use d
ni th is er act ion w asni ve st igate d. Mutl ipel er act ion swe er
con ducte d at 50 C for 24 h ni a solvent -fere syste m.
Atfe rthe compelt ion ofe ach run, the cat alyst w as ftile er d
an dw ashe dth ere t ime sw tih eth yl acet ate t o er move the
er sidual sub st rate s an dthe er act ion w as er pe ate d usni g
fer sh sub st rate s. As sh own ni F ig. 4, the content s of
isosobr ide an d caprylic acidwe er approximate lyze ro an d
be low 5 % er spe ct ive ly atfe r er use d1 6b at che s. Th is
de mon st rate s th at the con ve rsion s ni the se er act ion s
we er h ighe rth an 95%. The content s of mon oe ste r an d
dei ste rni the se cycel swe er b alan ce d at ab out 1 0% an d
80%. It is de mon st rate dth at the product w ate r can be
er act ion, an dth at the h ydrolysis of dei ste r is much e asei r
.
th an ti s synthe si 29
s
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA27537G
F ig. 5. TGA curve s of PVC b eln ds
is an e fef ct ive meth od fore ste rificat ion
.
30
Me ch an ical prope trei s of PVC b eln dswe er ch aracte rzie d
b y usni g E4 3.1 04 Un ive rsal Te st ni g Mach nie MTS
In st rument C rop., Ch ni a accordni gt o GB/T1 04 0.1 -
2006 Ch ni a un de r amb eint con dti ion s. Tab el
100
3
80
summarzie sthe value s ofthe me ch an ical prope trei s of
PVC b eln ds. The addti ion of isosobr ide dei ste rni the PVC
b eln ds cause dni cer ment ni ten seil st ern gth an der duct ion
ni e lon gat ion at b er ak. It is accordan ce w tih the common
ruel th at the plast iczie r can ni cer ase the e lon gat ion at
b er ak. The er ason maybe th at the compat bi itli ybetween
plast iczie r moel cular an d PVC moel cular is we ,ll so
isosobr ide dei ste r could be use d as an e fef ct ive
plast iczie r for PVC.
caprylic acid
Isosorbide
Monoester
Diester
60
40
20
0
0
2
4
6
8
10
12
14
16
Batches
Tab el 3. Me ch an ical prope trei s of PVC b eln ds.
F ig. 4. Re pet ti ive synthe sis of isosobr ide dicaprylate e ste r
b y Novez ym 4 35. Re act ion con dti ion s: 9. 855
Yei ld st er ss
Elon gat ion
rupt uer st er ss
e lon gat ion at
b er ak
g
isosobr ide, 1 9.44 g caprylic acid, 1. 97 g Novoz ym4 35, at
300 rpm for 24 h at 60 C.
1 6. 525 MPa
1 02.1 28 %
14. 66 MPa
236.7 04 %
maximum load
ten seil
st ern gth
large st ts rani
e lon gat ion
given e lon gat ion
st er ss
E. Prope trei s of plast iczie r on PVC
TGAw as use dt o st udythe the rmal de gradat ion of PVC
plast iczie dw tih I sosobr ide dei ste .r The paramete rs an d
curve s of TGA aer per sente dni F ig. 5. As sh own ni F ig.
1 06. 39 N
24. 06 MPa
227. 08 %
1 6. 36 Mpa
5, all ofthe PVC b eln dswe er the rmally st ab el ni
N
at mosphe er be low 90 C an dwe er divide dnit o ath ere2 -
F. Sub -ch ron ict oxicti y
st age the rmal de gradat ion
te mpe rat uer. The first st age de gradat ion at aroun d 90
260 C is corer spon dni g t o e vaporat ion an d
de composti ion ofw ate ,r HC l an d small moel cuel s. The
se con d st age at aroun d 260 4 52 C isthe faste st an d
couldbe att rbi ute dt othe format ion an d st oich iomet ric
e limni at ion of HC .l The last st age at ab ove 4 52 C is
att rbi ute dt o cross nlik ni g cont ani ni gC=C b on ds. The rmal
de gradat ion of polyene sni volve s cyczli at ion an d sptlit ni g
of ch ani s.
proce ss ab ove the
The saeft y of isosobr ide e ste rw ase valuate db yte st ni g
the sub -ch ron ict oxicti y ni mice w tih a 28-dayt er at ment
w tih difef ernt dose s of isosobr ide dei ste .r
1
Body an dbeh avior ob se rvat ion
As sh own ni Tab el 4, the appe aran ce, act ivtiei s, an d
psych osis of all the mice ni b oth isosobr ide dei ste r
t er at ment an d cont rol groupswe er alln orma,l an dthe er
we er n o abn ormal disch arge an d de ath. The er we er n o
sign ificant difef ern ce s ni mice b ody we ight between
cont rol an dt er at ment groups.
Isosorbide diester / PVC
100
2
He mat ologica,l live r an dk idne y an alysis
80
60
40
20
0
The an alysis ofb lood paramete rs iser el vant t o risk
e valuat ion be cause the ch an ge sni the he mat ological
syste mh ave ah ighe r per dict ive value forh uman t oxicti y
when the dat a aer t ran slate d from an imal st udei s
As sh own ni Tab el 5, at the en d ofthe e xpe riment
28 days , the WBC, R BC, HGB an d PL T sh owe dn o
sign ificant difef ern ce s between cont rol an d isosobr ide
dei ste r t er at ment
.
31
atfe r
0
100
200
300
400
500
600
groups. It is ni dicate d th at the
Temperature (
℃)
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isosobr ide dei ste r didn ot afef ct the he mat ological syste m
of mice.
Journal Name
DOI: 10.1039/C5RA27537G
The an alysis of live r an dk idne y aer sh own ni Tab el 6.
The er we er n o sign ificant difef ern ce sni live r an dk idne y
atfe rt er at ment w tih isosobr ide dei ste .r Atfe r 28 days
t er at ment w tih isosobr ide dei ste ,r the live r an dk idne y of
the t er at ment groupswe er almost the same w tih th ose of
the cont rol groups, illust rat ni gth at the isosobr ide dei ste r
w asn ot h armfult o mice live r an dk idne y.
3 O rgan h ist opath ology
As sh own ni F ig. 6, atfe r t er at ment w tih isosobr ide
dei ste ,rthe er we er n o sign ificant path ological ch an ge sni
live .r The live r ce llsni the live rt issue we er n ormal an dthe
live r lob uel swe er t idy an d compelte. Similarly, isosobr ide
dei ste rt er at ment didn t ni duce sign ificant ch an ge s ni
te st is of mice. The ch ambe r ofthe se mni ief roust ube
arran ge d er gularly, the w all ofthe b ase ment me mb rane
w as nite grti y, an dthe lumen swe er full of spe rms. In
summary, isosobr ide dei ste rt er at ment did n ot ni duce
sign ificant h ist opath ological ch an ge sni mice.
F ig.
st ruct uer of cont rol mice
isosobr ide dei ste rt er at ment mice
st ruct uer of cont rol mice H . D: the k idne y st ruct uer of
isosobr ide dei ste rt er at ment mice
6
. O rgan h ist opath ology ph ot ograph. A: the live r
H . B: the live r st ruct uer of
H . C: the k idne y
H .
Tab el 4. The b ody an dbeh avior ob se rvat ion of mice
mice
ef mael
appe aran ce
act ivtiei s
psych osis
abn ormal
disch arge
de ath
We ight
st atr
g
mael
3
en d
C ont rol
H
3
3
3
3
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
n one
1 9. 0
1 8. 3
1 9.1
30. 9
4 0. 8
32. 2
4 0. 5
32.4
C ont rol
H
C ont rol
M
C ont rol
M
3
3
3
1 9.7
C ont rol
L
1 8. 8
1 9. 2
1 9.4
C ont rol
L
4 0.7
31. 9
39. 6
Sampel
H
Sampel
H
1 9. 6
8 | J. Name., 2012, 00, 1-3
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ARTICLE
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Sampel
M
3
3
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n ormal
n one
n one
n one
n one
n one
n one
n one
n one
1 8. 6
1 8. 6
1 9. 0
1 9. 3
30. 3
4 0. 3
32. 9
Sampel
M
3
3
n ormal
n ormal
n ormal
Sampel
L
Sampel
L
4 0. 0
Tab el 5. He mat ological an alysis
mice
WBC
R BC
HGB
PL T
ef mael
mael
×1 0 L
×1 10
L
×gL
×1 0 L
*
9
1-
）
2
1-
）
1-
）
9
1-
）
*
*
*
（
（
（
（
C ont rol
C ont rol
C ont rol
C ont rol
H
H
M
M
3
3
3
3
3
3
5.4 ±0.7
5. 9±0. 8
5.7 ±1. 5
5. 9±0.4
5. 5±0. 5
5. 8±0. 3
4.7 ±0. 5
6. 2±0.7
14 2. 0±14.7
1 34. 0±1 3. 9
1 34.7 ±1 3. 6
1 33. 3±1 0. 3
14 2. 3±6.7
1 82. 0±36.7
17 5. 6±29. 9
1 51.7 ±26.7
174.7 ±1 5. 6
17 8. 3±1 2. 0
17 3. 6±8. 6
1 67. 0±1 6. 6
17 2. 6±1 6. 6
1 61. 0±14. 2
1 57. 0±2. 6
1 55. 3±17. 6
1 53.7 ±1 6. 9
3
3
3
3
3
3
C ont rol L
C ont rol L
5.7 ±0. 6
5. 9±0. 5
5.1 ±0.7
4. 6±0. 6
5. 9±0. 2
5. 8±0.4
1 30. 0±2. 0
1 35. 6±14.4
1 34. 0±11. 5
1 35. 3±1 2.7
1 33. 0±1 5.7
1 29.7 ±2. 5
1 31.7 ±6.1
Sampel
Sampel
Sampel
Sampel
Sampel
Sampel
H
H
M
M
6. 0±1.7
5.1 ±0. 8
5. 5±0. 5
6.7 ±1. 2
6. 5±0. 5
5. 8±0. 3
4. 8±0. 3
4. 5±0. 9
5. 3±0. 6
5.1 ±0. 6
L
L
Tab el 6. L ive r an dk idne y an alysis
AL T
U L/
BU N
mmolL/
PT
S
APTT
TT
S
FI B
gL/
S
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C ont rol
C ont rol
C ont rol
C ont rol
H
H
M
M
24. 0±7. 5
26. 3±4. 0
1 9. 3±3. 8
25.1 ±6. 6
25. 3±8. 5
25.7 ±6. 0
26. 0±7. 8
23. 3±1. 5
22. 3±2. 5
25. 0±5. 0
24. 3±5.1
6. 6±0. 8
6.4 ±1. 2
5. 9±1. 2
4. 9±1. 0
6.1 ±0. 8
5. 9±0. 6
5. 3±1. 5
6. 0±1. 0
5.4 ±0. 5
5. 3±1. 5
5. 6±0. 5
6. 3±0. 6
1 0.7 ±0. 6
11.1 ±1. 2
1 0. 8±1. 0
11.7 ±0.4
27. 3±2. 5
25. 6±3. 0
26. 9±3.7
26. 3±2.4
26. 5±2. 2
27. 3±1.4
27. 2±1.7
26. 8±1. 0
26.7 ±1.1
26. 6±1. 3
26. 8±2. 0
28. 2±1. 2
1 6.1 ±1. 2
1 6. 9±1. 0
17.4 ±3. 2
1 6. 2±1.7
2.4 ±0. 5
2. 3±0. 3
2. 2±0. 2
2. 3±0. 3
2. 2±0. 3
2.4 ±0.4
C ont rol L
C ont rol L
11. 2±1. 0
11. 5±0. 5
1 0. 9±0. 6
11.7 ±0.4
17. 2±1. 3
1 6. 2±0. 8
1 6. 9±1. 0
1 6. 6±0. 6
1 6. 6±1. 5
1 6. 3±2. 3
17. 5±0. 5
17. 0±1. 0
Sampel
Sampel
Sampel
Sampel
Sampel
Sampel
H
2.1 ±0. 3
2. 3±0.4
H
M
M
11. 5±0. 5
11. 0±1. 0
11.1 ±1. 2
11. 3±0. 8
2. 5±0. 2
2.4 ±0. 6
2. 3±0. 5
2. 0±0.1
L
L
21.7 ±3. 8
Conclusions
Notes and references
Nat ion al Ene rgyR& DCente r for Bioer fnie r,y Be inji g Ke yL ab orat ory
of Bioproce s,sC oell ge of Biology Scein ce an d Te chn ology, Be inji g
In th is st udy, isosobr ide dei ste rw as synthe szie dw tih
middel ch ani caprylic acid un de r solvent -fere con dti ion s
w tih low el ve ls of mon oe ste r format ion. The crti ical
a
Un ive rsti y ofChe mical Te chn ology, Be inji g1 00029, PR Ch ni a. F ax:
+861 0 6441 6691 ; E-ma:il chenb qi ian g@ gma.il com;
t antw@ ma.ilb uct.e du. cn
paramete rs ni the
proce ss of e ste rificat ion we er
Sh an don g Jqi ni gChe micaCl o., Lt d, Sh an don g, 262500, PR Ch ni a.
E-ma:ilzhen yue ju0033@1 63. com.
b
discusse d an dthe opt imal con dti ion swe er dete rmnie d.
Base d on act ivat ion ene rgy an alysis, ti w as dete rmnie d
th at synthe szi ni g dei ste r from the mon oe ste r is the
limtini g ste p ni the synthe sis. The discove ry of the
limtini g ste p is sign ificant for ni dust rial product ion. The
prope tr y of isosobr ide dei ste r as plast iczie r on PVC w as
dete rmnie d. F utrhe rmoer, the sub -ch ron ic t oxicti y of
isosobr ide dei ste rw aste ste d ni mice, an dthe er sutl s
sh owe d th at the dei ste r w as b iosaef. So isosobr ide
dei ste rh as ger at potent ial forni dust rial applicat ion as a
plat iczie .r
De patr ment of Biology an d Ph armacy, Zhe jian g Ph armace ut ical
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ARTICLE
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Graphic
Highlight
The biosafety isosorbide dicaprylate ester plasticizer was synthesized
with bubbling dried air in solvent-free system