Lipase-catalyzed transformations using poly(ethylene glycol) as solvent

Article abstract of DOI:10.3109/10242422.2011.578739

Candida antarctica lipase catalyzes a number of elementary reactions like alcoholysis, ammoniolysis and aminolysis in poly(ethylene glycol) (PEG) media. Reaction rates were comparable to or better than those observed in conventional organic reaction media and ionic liquids. It is envisaged that PEGs could have added benefits for performing biotransformations with highly polar substrates, which are sparingly soluble in common organic solvents.

Full text of DOI:10.3109/10242422.2011.578739

Biocatalysis and Biotransformation, July–August 2011; 29(4): 113–118
ORIGINAL ARTICLE
Lipase-catalyzed transformations using poly(ethylene glycol)
as solvent
MAZAAHIR KIDWAI, ROONA PODDAR & SAURAV BHARDWAJ
Green Research Laboratory, Department of Chemistry, University of Delhi, Delhi, India
Abstract
Candida antarctica lipase catalyzes a number of elementary reactions like alcoholysis, ammoniolysis and aminolysis in
poly(ethylene glycol) (PEG) media. Reaction rates were comparable to or better than those observed in conventional
organic reaction media and ionic liquids. It is envisaged that PEGs could have added benefits for performing biotransfor-
mations with highly polar substrates, which are sparingly soluble in common organic solvents.
Keywords: Lipase, polyethylene glycol, heterogeneous catalyst, esterification, ammoniolysis, aminolysis
Introduction
position products make their utility limited (Kamal
Following the pioneering studies of Klibanov and
co-workers, the use of hydrolytic enzymes in anhy-
drous organic media has become a valuable addition
to the synthetic repertoire (Zaks & Klibanov 1984).
Reactions which are impossible in water can be eas-
ily performed and enzymes can even show enhanced
thermostability (Zaks & Klibanov 1985; Klibanov
1986) in such media. Numerous methods have been
reported for the activation of lipases in non-aqueous
media such as lipid-coating-mediated activation,
entrapment of lipases in hydrophobic sol–gel materi-
als, molecular bioimprinting, salt-mediated activation
and the use of some surfactants e.g. polyoxyethylene
alkyl ether (Abe et al. 2008). However, a serious draw-
back of conventional organic media is the substantial
reduction in rate of reaction, which is caused mainly
by altered partitioning of the reactant between the
solvent and the active site (Das et al. 2008; Shen
et al. 2008;Vidya & Chadha 2009). Moreover, envi-
ronmental problems are associated with the use of
many volatile organic solvents like chlorinated hydro-
carbons. Ionic liquids, especially imidazolium-based
systems containing PF6Ϫ and BF4Ϫ anions, are toxic
in nature as they liberate hazardous HF; moreover
their high cost and problems of their final disposal
following the gradual accumulation of side or decom-
et al. 2005; Jain et al. 2007). Besides their environ-
mental effects, some ionic liquids especially 1-butyl-
3-methylimidazolium nitrate ([bmim][NO3]) can also
denature the free enzyme (Candida antarctica lipase B
cross-linked on a polypropylene) (Toral et al. 2007).
Poly(ethylene glycol) (PEG) has been success-
fully used as an alternative reagent to replace ionic
liquids and organic solvents in biphasic catalytic
systems. PEG homopolymers present some interest-
ing characteristics, including high polarity and high
boiling points and special properties arising from
their cation complexation ability. They have been
used in substitution, oxidation, reduction and orga-
nometallic reactions. Furthermore they present low
toxicity and have been used for biomedical applica-
tions, especially in the field of drug delivery, and as
soluble polymeric supports in organic synthesis. PEGs
with low molecular weight (less than 800 Da) are liquid
at room temperature, while higher-molecular-weight
PEGs are solids that melt at a moderate temperature
and can therefore be used as solvents (Colacino et al.
2007). As an alternative to ionic liquids, Reetz and
Wiesenhofer (2004) demonstrated that the combina-
tion of PEG and supercritical carbon dioxide consti-
tutes a cheap and benign biphasic solvent system for
enzymatic reactions. They used PEG 1500 which is
Correspondence: M. Kidwai, Green Research Laboratory, Department of Chemistry, University of Delhi, Delhi – 110007, India. Tel/fax: ϩ91-11-2766635.
E-mail: kidwai.chemistry@gmail.com
(Received 31 December 2010; revised 13 March 2011; accepted 4 April 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.578739

114
M. Kidwai et al.
a solid at room temperature and thus leads to a
multiphase solvent system (Reetz et al. 2004). To
overcome this situation, PEG 200, 400 and 600 were
used in the present work.
In continuation of our studies to develop improved
and environmentally benign methodologies for organic
reactions (Kidwai et al. 2009a, Kidwai et al. 2002), we
describe herein the combination of C. antarctica lipase
and PEG as a reaction medium to catalyze a number
of elementary reactions under neutral reaction condi-
tions. To verify the action of lipase, long-chain fatty
acids were used as substrates in these reactions.
and shaken at room temperature. Lipase was then
added to start the reaction. The progress of the reac-
tion was monitored by TLC as well as HPLC. In the
TLC of crude reaction mixtures, two spots were
observed:onefortheacidwithretentiontimeRf ϭ 0.88
and another for the amide with Rf ϭ 0.82/ester with
Rf ϭ 0.95. After completion of the reaction, diethyl
ether (10 mL) was added to the reaction mixture and
a dry ice–acetone bath was used to solidify the PEG.
PEG was allowed to settle to the bottom and the prod-
uct partitioned into the diethyl ether layer, which was
decanted off.The diethyl ether layer was washed with
5% w/v sodium bicarbonate solution (to remove acid),
1 M hydrochloric acid (to remove amine) and brine
(Vandevoorde et al. 2003).The organic layer was dried
over anhydrous sodium sulfate and concentrated under
reduced pressure. The structures of all products were
established unambiguously on the basis of their spec-
Materials and methods
General
1H-NMR and 13C-NMR were spectra recorded on a
Bruker TOP SPIN 300 MHz and 75.6 MHz spectro-
meter, (United States, North America) with chemical
shift values (δ) in ppm downfield from TMS using
CDCl3 as solvent. IR spectra of the samples were
recorded on a Perkin–Elmer FTIR-1710 spectrometer
(Massachusetts, USA) using KBr. Elemental analyses
were performed using a Heraeus CHN-Rapid Analyzer
(London, UK). EI mass spectra were recorded on a
time-of-flight mass spectrometer. Melting points were
determined on aThomas Hoover melting point appa-
ratus (Thomas Scientific, USA) and are uncorrected.
The purity of compounds was checked on silica-
gel-coated aluminum plates (product number
1.05554.0007) (Merck TLC; particle size 10–12 μm,
particle distribution 5–20 μm, layer thickness 250 μm,
plate height 30 μm). HPLC used a system consisting
of a CLC-ODS (M) column, an RID-2AS refractive
index detector and an LC-4A integrator (Shimadzu).
1
tral analysis (IR, H- and 13C-NMR and GC/MS
data). All products were known compounds and there
was no deleterious effect observed in scaling up the
reaction to 0.1 mol substrate.
Analysis of reaction mixtures
The course of enzymatic reaction was studied using
HPLC. At predetermined reaction times, 0.1 mL
aliquots were taken out and diluted with methanol–
chloroform (50:50, v/v). The immobilized enzyme
was removed from the reaction mixture by filtration.
Response calibration was done using authentic prod-
ucts. The mobile phase, retention times and flow
rates used for quantification of the different products
are shown in Table I.
Results and discussion
Chemicals
To examine the activity of lipase in different solvent
systems, the same reaction was carried out in an
organic solvent (i.e. t-BuOH), two ionic liquids and
three PEGs of different molecular masses. t-BuOH
was used as the organic solvent because it signifi-
cantly affects the mechanism, presumably by causing
conformational changes in the enzyme structure,
which also eliminates inhibition by alcohol. t-BuOH
increases the solubility of alcohol, eliminating its
inhibitory effect on lipases which can deactivate the
enzymes by denaturation (Türkan & Kalay 2008).
Two different ionic liquids, 1-butyl-3-methylimida-
zolium hexafluorophosphate ([bmim][PF6]) and 1-
butyl-3-methylimidazoliumtetrafluoroborate([bmim]
[BF4]), were used for the reaction.
C.antarctica lipase B (CALB;Novozym® 435) immo-
bilized on macroporous acrylic resin, a non-specific
lipase, was a gift from Novozymes, Bagsvaerd, Den-
mark. Molecular sieves sized 4 Å were supplied by
SpectroChem (India). Lauric acid (Ͼ98.5% pure),
capric acid (Ͼ95.5% pure), myristic acid (Ͼ98.5%
pure) and palmitic acid (Ͼ98.5%) were purchased
from laboratory in India. Solvents of HPLC quality
were supplied by SpectroChem (India).
Biotransformation procedures
Carboxylic acid (1 mmol) and ethanol (1 mmol)–
ammonia (2 mL)–amine (1 mmol) in PEG solvent
(2 mL) were mixed in a 10-mL round-bottomed flask,
and to this 100 mg of molecular sieves were added
To confirm the need for CALB, a blank reaction
was carried out in all three cases, confirming that
no product was formed in the absence of lipase.

Synergism between lipase and PEG 115
Table I. Mobile phases, retention time and flow rates for HPLC
analyses of the compounds.
(due to its large size) (Berg et al. 1998); and (2) the
weak nucleophilicity of PEG towards acid as
compared with alcohol. However, in the absence of
alcohol, PEG itself works as a substrate for lipase but
with poor yield. The small amounts of PEG ester/
PEG amide formed cannot be isolated from PEG as
they behave in an identical manner.The crown ether,
triglyme (triethylene glycol dimethyl ether), was also
tested as a solvent as it behaves like a small PEG
(molecular weight 180 Da), but this gave average
yields compared with t-BuOH.
After completion of the reaction and recovery of
the product, small amounts of unreacted acid and
alcohol/amine/ammonia and trace amounts of PEG
ester/PEG amide are in the reaction mixture. Alcohol/
amine/ammonia and acid can be removed in the reac-
tion workup, while PEG ester/PEG amide goes into
the PEG fraction. All conversions were confirmed by
HPLC after purifying the reaction mixture.
The enzymatic esterification was further exam-
ined with different fatty acids and ethanol using
CALB as catalyst in t-BuOH, the ionic liquids
[bmim][BF4] and [bmim][PF6], and PEG 200, 400
and 600. PEG, as a reaction solvent, promoted the
esterification in all cases. Presumably, the greater
stability of the transition state in PEG than in
t-BuOH is due to the high molecular weight of PEG,
and increases with increasing PEG molecular weight
(Table II). The enhancement in the rate of reaction
and yield in ionic liquid may be due to the fact that
the imidazolium cation might interact with charged
groups of the enzyme, either in the active site or at
Mobile phase Retention
Sample
no.
(methanol
–water, v/v)
time, Rf
(min)
Flow rate
(mL min–1)
Compound
1
2
3
4
5
6
7
8
Capramide
85:15
98:2
85:15
98:2
90:10
98:2
95:5
100:0
100:0
98/2
100/0
100/0
10.2
5.2
15.6
5.4
12.8
4.4
5.8
5.2
3.2
6
0.5
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Ethyl caprate
Capric acid
Ethyl laurate
Lauramide
Lauric acid
Myristamide
Myristic acid
Ethyl myristate
Palmitamide
Palmitic acid
Ethyl palmitate
9
10
11
12
6.4
3.4
Additionally, to investigate whether PEG had any
catalytic activity, a similar set of reactions were per-
formed in PEG 600 as solvent without lipase. In this
case, a trace amount of product was formed in the
absence of lipase due to a side reaction. Subse-
quently, we carried out the same reactions using
PEG, ionic liquids and t-BuOH as solvent in the
presence of lipase and excellent yields were obtained
with both PEGs and ionic liquids, showing a syner-
gism between lipase and PEG (Figure 1). Low-
molecular-weight liquid PEGs can be regarded as
protic solvents with aprotic binding sites comprised
of the ethylene oxide units (Heinsman et al. 2001).
However, in the presence of water, PEG can also
work as a substrate for lipase (Heinsman et al. 2001).
Thus molecular sieves were used to absorb the water
from the reaction mixture so that the PEG could not
act as a lipase substrate (Lau et al. 2000).The forma-
tion of the mono-alcohol esters/amides rather than
PEG ester/PEG amide is attributed to the following:
(1) the active site of lipase is composed of two dis-
tinct pockets and buried under a helical lid, thereby
making it inaccessible to external solvent and PEG
Table II. Esterification of fatty acids with ethanol catalyzed by
immobilized CALB (Novozym 435)a.
O
O
R
OH
+
R
OC2H5
Time (min)
C2H5OH
Solvent
R
Conversion (%)
t-BuOH
C11H23
C11H23
C11H23
C11H23
C11H23
C9H19
C13H27
C15H31
C11H23
C9H19
C13H27
C15H31
C11H23
C9H19
C13H27
C15H31
43
87
79
44
77
62
81
82
89
86
88
91
90
86
92
92
30
30
30
30
30
35
25
25
30
35
25
25
30
35
25
25
[bmim][PF6]
[bmim][BF4]
Triglyme
PEG 200
PEG 400
PEG 600
Figure 1. Time course of the condensation of lauric acid and
ethanol catalyzed by immobilized CALB (Novozym 435). Reaction
conditions: 1 mmol lauric acid and 1 mmol ethanol in 2 mL
solvent and 25 mg enzyme were stirred at room temperature.
aReaction conditions: 1 mmol fatty acid, 1 mmol ethanol and
25 mg enzyme in 2 mL solvent were stirred at room temperature.

116
M. Kidwai et al.
its periphery, causing changes in the enzyme struc-
ture (Kidwai & Poddar 2008). A somewhat lower
reaction rate in t-BuOH can be ascribed to inhibition
by the solvent (Figure 1).
It has been shown that the condensation of a car-
boxylic acid and ammonia in the presence of a lipase
is feasible, albeit with very long reaction times; for
example 90–100% conversion in 17 days using ammo-
nium carbamate in methylisobutyl ketone (Litjens et
al. 1999). The reaction of lauric acid with ammonia
was carried out in ionic liquid in the presence of
CALB, by bubbling ammonia into the reaction. A
quantitative conversion was obtained after 2–3 days.
We compared the ammoniolysis of lauric acid in PEG
medium with that in ionic liquid and t-BuOH under
identical reaction conditions and obtained similar
conversions (Table III). The increased yield in PEG
and ionic liquid can be explained on the basis that
both can trap more ammonia than t-BuOH. The
kinetics of amidation using free acids is known to be
controlled by the solubility of the ion pair formed by
the reactants. Moreover it has been found that the
selectivity of the reaction depends on the solubility of
the product in the solvent used and that the choice of
solvent is critical in obtaining an efficient process (de
Zoete et al. 2006).
Figure 2. Time course of the condensation of lauric acid and
amine catalyzed by CALB (Novozym 435) in PEG 600. Reaction
conditions: 1 mmol lauric acid and 1 mmol ethyl amine in 2 mL
PEG 600 and 25 mg enzyme were stirred at room temperature.
was 82%. In the ionic liquids [bmim][BF4] and
[bmim][PF6], it was 77% and 73% respectively, while
in t-BuOH this fell to 65% (Figure 2 and Table IV).
Table IV. Aminolysis of fatty acids with amines, catalyzed by
immobilized CALB (Novozym 435)a.
O
O
R
NR1H
+ NR1H2
R
OH
Solvent
R
R1
Conversion (%) Time (h)
N-substituted fatty acid amides are important
commodities which can be easily prepared under mild
conditions via lipase-catalyzed reaction of a carboxylic
ester and an amine (Kidwai et al. 2009b). The yield
of N-ethyl lauric acid amide obtained in PEG 600
was 88%, in PEG 400 was 85% and in PEG 200
t-BuOH
C11H23
C11H23
C11H23
C11H23
C11H23
C9H19
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
65
72
73
79
70
77
69
71
72
82
60
67
66
70
69
83
74
85
60
70
69
77
76
85
78
88
66
75
73
79
75
89
10
7.5
8
5
8
5
8
8
8.5
6
10
7
7.5
5.5
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
[bmim][PF6]
[bmim][BF4]
Triglyme
Table III. Ammoniolysis of fatty acids with ammonia catalyzed by
immobilized CALB (Novozym 435)a.
PEG 200
O
O
R
NH2
C13H27
C15H31
C11H23
C9H19
NH3
R
OH
+
Solvent
R
Conversion (%)
Time (h)
t-BuOH
C11H23
C11H23
C11H23
C11H23
C11H23
C9H19
C13H27
C15H31
C11H23
C9H19
C13H27
C15H31
C11H23
C9H19
C13H27
C15H31
56
83
61
58
68
61
72
75
71
63
76
78
72
65
75
78
96
96
96
96
72
96
72
72
96
96
72
72
72
96
72
72
PEG 400
[bmim][PF6]
[bmim][BF4]
Triglyme
PEG 200
C13H27
C15H31
C11H23
C9H19
PEG 400
PEG 600
PEG 600
C13H27
C15H31
4
aReaction conditions: 1 mmol fatty acid, 2 mL ammonia and
25 mg enzyme in 2 mL solvent were stirred at room temperature.
aReaction conditions: 1 mmol fatty acid, 1 mmol amine and 25 mg
enzyme in 2 mL solvent were stirred at room temperature.

Synergism between lipase and PEG 117
References
Table V. Reusability of immobilized CALB (Novozyme 435) in a
4 h reactiona.
AbeY, Hirakawa T, Nakajima S, Okano N, Hayase S, Kawatsura
M, HiroseY, ItohaT. 2008. Remarkable activation of an enzyme
by (R)-pyrrolidine-substituted imidazolium alkyl PEG sulfate.
Adv Synth Catal 350:1954–1958.
Berg OG, Cajal Y, Butterfoss GL, Grey RL, Alsina MA,Yu B-Z,
Jain MK. 1998. Interfacial activation of triglyceride lipase from
Thermomyces (Humicola) lanuginosa: kinetic parameters and a
basis for control of the lid. Biochemistry 37:6615–6627.
Colacino E, Daïch L, Martinez J, Lamaty F. 2007. Microwave-
assisted copper-catalyzed sonogashira reaction in PEG solvent.
Synlett 1279–1283.
Das S, Chandrasekhar S, Yadav JS, Rao AVR, Grée R. 2008.
An efficient process for the resolution of cis-4-O-protected-2-
cyclopenten-1,4-diol using pancreatin lipase in [C8mim][PF6]
as a reusable system. Tetrahedron Asymm 19:2543–2545.
de Zoete MC, Kock-van Dalen AC, Rantwijk F, Sheldon RA.
1996. A new enzymatic one-pot procedure for the synthesis of
carboxylic amides from carboxylic acids. J Mol Catal B: Enzym
2:19–25.
Sample no.
Enzyme run
Yield (%)
1
2
3
4
5
6
7
8
Fresh
I
II
88
88
88
87
87
86
85
83
76
72
67
62
III
IV
V
VI
VII
VIII
IX
X
9
10
11
12
XI
aReaction conditions: 1 mmol lauric acid, 1 mmol ethylamine and
25 mg enzyme in 2 mL PEG 600 were stirred at room
temperature.
Heinsman NWJT,Teixeira A.Van DerWeide PLJ, Franssen MCR,
Van Der Padt A, De Groot A, Riet KV. 2001. Esterification of
4-methyloctanoic acid with polyethylene glycol at different aw.
Biocatal Biotransformation 19:181–189.
Jain SL, Singhal S, Sain B. 2007. PEG-assisted solvent and cata-
lyst free synthesis of 3,4-dihydropyrimidinones under mild
reaction conditions. Green Chem 9:740–741.
Kamal A, Reddy DR, Rajendar. 2005. A simple and green proce-
dure for the conjugate addition of thiols to conjugated alkenes
employing polyethylene glycol (PEG) as an efficient recyclable
medium. Tetrahedron Lett 46:7951–7953.
Kidwai M, Poddar R. 2008. Transesterification of chromenes
employing immobilized lipase in ionic liquids. Catal Lett
124:311–317.
Kidwai M, Dave B, Bhushan KR, Misra P, Saxena RK, Gupta R,
Gulati R, Singh M. 2002. Deacylation of cephalosporins by
lipase catalysis and microwave assisted transformation on a
solid support. Biocatal Biotransformation 20:377–379.
Kidwai M, Poddar R, Diwaniyan S, Kuhad RC. 2009a. Laccase
from basidiomycetous fungus catalyzes the synthesis of substi-
tuted 5-deaza-10-oxaflavins via a domino reaction. Adv Synth
Catal 351:589–595.
Any biocatalyst reuse obviously impacts upon
process economics. A study was performed to
determine the enzyme activity with repeated reuse.
The reusability of the enzyme was examined by
following repeated rounds of aminolysis with lauric
acid and ethylamine under optimized conditions in
PEG 600 for 4 h, as described above. Between
catalytic cycles the enzyme was filtered, washed
with hot 1,4-dioxane and dried in air at room
temperature, then reused. There was a marginal
decrease in activity by runVII, which might be due
to handling, but after that there was a more sub-
stantial and sustained decrease in the lipase activity
(Table V).
Conclusions
Kidwai M,Poddar R,Mothsra P.2009b.N-acylation of ethanolamine
using lipase: a chemoselective catalyst. Beil J Org Chem 5:10.
Klibanov AM. 1986. Enzymes that work in organic solvent.
Chemtech 16, 354–359.
Lau RM, Rantwijk FV, Seddon KR, Sheldon RA. 2000. Lipase-
catalyzed reactions in ionic liquids. Org Lett 2:4189–4191.
Litjens MJJ, Straathof AJJ, Jongejan JA, Heijnen JJ. 1999. Synthe-
sis of primary amides by lipase-catalyzed amidation of carbox-
ylic acids with ammonium salts in an organic solvent. Chem
Commun 1255–1256.
C. antarctica lipase was shown to catalyze alcoho-
lysis, ammoniolysis and aminolysis reactions using
PEGs of molecular weight 200, 400 and 600 Da
as reaction media. Reaction rates were generally
comparable with, or better than, those observed
in ionic liquids and organic media. There is a
synergism between enzyme catalysis and PEG as
reaction media.
Reetz MT,WiesenhoferW. 2004. Liquid poly(ethylene glycol) and
supercritical carbon dioxide as a biphasic solvent system for
lipase-catalyzed esterification. Chem Commun 2750–2751.
Shen ZL, Zhou WJ, LiuYT, Ji SJ, Loh TP. 2008. One-pot chem-
oenzymatic syntheses of enantiomerically enriched O-acetyl
cyanohydrins from aldehydes in ionic liquid. Green Chem
10:291–294.
Toral AR, De los Ros AP, Hernandez FJ, Janssen MHA, Schoe-
vaart R, Rantwijk F, Sheldon RA. 2007. Cross-linked Candida
antarctica lipase B is active in denaturing ionic liquids. Enzyme
Microb Technol 40:1095–1099.
Acknowledgements
R. Poddar and S. Bhardwaj are grateful to CSIR
New Delhi for providing finance assistance and
M. Kidwai is grateful to the University of Delhi for
providing finance assistance.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Türkan A, Kalay . 2008. Study of the mechanism of lipase-
catalyzed methanolysis of sunflower oil in tert-butanol and
heptane. Turk J Biochem 33:45–49.

118
M. Kidwai et al.
Vandevoorde S, Jonsson KO, Fowler CJ, Lambert DM. 2003.
Modifications of the ethanolamine head in N-palmitoyleth-
anolamine: synthesis and evaluation of new agents interfering with
the metabolism of anandamide. J Med Chem 46:1440–1448.
Vidya P, Chadha A. 2009. The role of different anions in
ionic liquids on Pseudomonas cepacia lipase catalyzed trans-
esterification and hydrolysis. J Mol Catal B: Enzym 57:
145–148.
Zaks A, Klibanov AM. 1984. Enzymatic catalysis in organic media
at 100°C. Science 224:1249–1251.
Zaks A, Klibanov AM. 1985. Enzyme-catalyzed processes in
organic solvents. Proc Natl Acad Sci U S A 82:3192–3196.
Supplementary materials available online