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Chemo-enzymatic route to synthesis of biodegradable polymers and glycolipid analogs

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Title:
Chemo-enzymatic route to synthesis of biodegradable polymers and glycolipid analogs
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Book
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English
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Bhatt, Surbhi
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University of South Florida
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Subjects / Keywords:
Lipase
Sophorolipid
Ionic liquid
Caprolactone
Maltose
Dissertations, Academic -- Chemistry -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: New catalytic synthetic methods in organic chemistry that satisfy increasingly stringent environmental constraints are in great demand by the pharmaceutical and chemical industries. Studies over last 15 years have revealed that activity of enzymes can be increased in organic solvents rather than their natural aqueous environment. Because of their ease of use, high selectivity and environment friendliness, enzymes are enjoying increasing popularity in today's synthesis world. Chapter 1 describes chemo-enzymatic synthesis of various glycolipid analogs. A highly regioselective macrolactonization was achieved using lipase from Candida antarctica as a catalyst. It also describes evaluation of lipases from different source and their efficiency in catalyzing the macrolactonization reaction. These analogs were synthesized using commercially available agriculture based disaccharides (maltose, lactose, cellobiose, melibiose). These glycolipid analogues have potential applications in the cosmetic industry, formulation, food production, and pharmaceutical industry.In Chapter 2, ring opening polymerization of epsilon-caprolactone in ionic liquid, bmimPF6 was investigated. A comparative study of ROP in different solvents (toluene, Ionic liquid, and bulk condition) was conducted. Effect of time and enzyme concentration on molecular weight and % yield was investigated. It was concluded that enzymatic ring opening polymerization of epsilon-caprolactone in ionic liquid, bmimPF6 is a very competitive and environmental friendly way of synthesizing high molecular weight polyesters.
Thesis:
Thesis (M.A.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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by Surbhi Bhatt.
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Title from PDF of title page.
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Document formatted into pages; contains 60 pages.

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ABSTRACT: New catalytic synthetic methods in organic chemistry that satisfy increasingly stringent environmental constraints are in great demand by the pharmaceutical and chemical industries. Studies over last 15 years have revealed that activity of enzymes can be increased in organic solvents rather than their natural aqueous environment. Because of their ease of use, high selectivity and environment friendliness, enzymes are enjoying increasing popularity in today's synthesis world. Chapter 1 describes chemo-enzymatic synthesis of various glycolipid analogs. A highly regioselective macrolactonization was achieved using lipase from Candida antarctica as a catalyst. It also describes evaluation of lipases from different source and their efficiency in catalyzing the macrolactonization reaction. These analogs were synthesized using commercially available agriculture based disaccharides (maltose, lactose, cellobiose, melibiose). These glycolipid analogues have potential applications in the cosmetic industry, formulation, food production, and pharmaceutical industry.In Chapter 2, ring opening polymerization of epsilon-caprolactone in ionic liquid, [bmim][PF6] was investigated. A comparative study of ROP in different solvents (toluene, Ionic liquid, and bulk condition) was conducted. Effect of time and enzyme concentration on molecular weight and % yield was investigated. It was concluded that enzymatic ring opening polymerization of epsilon-caprolactone in ionic liquid, [bmim][PF6 ] is a very competitive and environmental friendly way of synthesizing high molecular weight polyesters.
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Chemo-Enzymatic Route to Synthesis of Biodegradable Polymers and Glycolipid Analogs by Surbhi Bhatt A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Kirpal S. Bisht, Ph.D. Abdul Malik, Ph.D. Edward Turos, Ph.D. Date of Approval: April 7, 2006 Keywords: lipase, sophorolipid, io nic liquid, caprolactone, maltose Copyright 2006 Surbhi Bhatt

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ACKNOWLEDGEMENTS More than can be expressed in words, I am grateful to my ma jor professor Dr. Kirpal S. Bisht whose guidance and encouragement has been invaluable throughout my graduate studies and preparation of this thesis. Appr eciation is extended to the members of my committee Dr. Edward Turos and Dr. Abdul Ma lik for their guidance during my graduate studies. I would like to thank all of my gr oup members, Mr. Pasha M. Khan, Mr. R. Wu, Mr. Sumedh Parulekar, Mr. Sr idhar Reddy Kaulagari and Ms Ki ran K. Mupalla, for their assistance and support. I would like to thank Dr. Ted Gauthier for his help with MALDI analysis and Dr. Edwin Rivera for NMR anal ysis. I appreciate constant support of my husband, Sanjeev Sati, throughout my graduate studies. Financial support from USF Department of Sponsored Research, the Amer ican Cancer Society, the American Lung Association, American Cancer Society IRG program and the Frasch Foundation is greatly appreciated.

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i Table of contents List of Tables and Charts ii List of Figures iii List of Schemes iv List of Symbols and Abbreviation v Abstracts vi Chapter 1: Lipase-Catalyzed Synthesis of Glycolipid analogs 1 1.1 Introduction 1 1.2 Bio-activity of Glycolipids 3 1.3 Traditional syntheses 4 1.4 Experimental section 10 1.4.1 Chemicals and enzymes 10 1.4.2 Column chromatography 10 1.4.3 Nuclear magnetic resonance 11 1.4.4 General synthesis of octaacetate of lactose maltose, melibiose and cellobiose 11 1.4.5 Synthesis of methyl 15-hrdroxypentadecanoate 11 1.4.6 Synthesis of compound 3a-d 12 1.4.7 Synthesis of compoubd 4a-d 13 1.4.8 Screening of lipase and genera l procedure of lipase catalyzed macrolactonization and lipase catalyzed ester hydrolysis 13 1.5 Result and Discussion 14 1.5.1 Screening of Lipase 19 1.5.2 Lipase-catalyzed macrolac tonization 20 1.5.3 Lipase catalyzed hydrolysis of the methyl ester 21 1.5.4 Structure elucidation of macrolactone lipids 5a and 5b 23 1.6 Conclusion 27 1.7 Reference 28 Chapter 2: Ring opening polymerization of -Caprolactone in ionic liquid 2.1 Introduction 29 2.2 Experimental Section 34 2.2.1 Procedure for polymerization 34 2.2.2 Preparation of 1-buty l-3-methyl imidazolium Hexaflourophosphate 34 2.2.3 Molecular weight Measurements 35 2.3 Results and Discussion 36 2.4 Conclusion 43 2.5 References 44 Appendix 47

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ii List of Tables and Charts Chart 1.1 Flow chart for fermentation process 5 Chart 1.2 Flow chart of the coupled pro cess including filtration 6 Table 2.1 Comparison of polymeriza tion in different mediums with monomer:enzyme:solvent ratio being 2:1:2 by weight at 75 oC for 40 h. 38 Table 2.2 Comparison of polymeriza tion in different mediums with monomer:enzyme:solvent ratio being 4:1:4 by weight at 75 oC 38 Table 2.3 Time-related study of polym erization in ionic liquid at 65 oC 39 Chart 2.1 Plot of molecula r weight as function of time 41 Chart 2.2 Plot of % yield as a func tion of time 42

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iii List of Figures Figure 1.1 Various natural Glycolipids 2 Figure 1.2 Different glycolipid analogs 14 Figure 1.3 Different disaccharide used as starting material 15 Figure 1.4 DEPT showing lipase catalyzed hydr olysis of methyl ester 23 Figure 1.5 DEPT-135 showing lipas e catalyzed macrolactonization at C6 position 25 Figure 1.6 HMBC of product 5a showing correlation between C-6 and C-1 26

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ivList of Schemes Scheme 1.1 Retro-synthesis of a Tricolorin A intermediate by Furstner et al 8 Scheme 1.2 Retrosynthesis of Tricolor in-A by Hitchcock et. al 9 Scheme 1.3 Synthetic approach to diffe rent glycolipid analogs 16 Scheme 1.4 Synthesis of 15-hydroxy pentadecanoate 16 Scheme 1.5 Acetylation of disaccharide 16 Scheme 1.6 Synthesis of 4a-d 18 Scheme 1.7 Screening different lipases for the regioselective macrol actonization of substrate 4a 19 Scheme 1.8 Lipase catalyzed macrolactonization 21 Scheme 1.9 Lipase catalyzed hydrolys is of methyl ester 22 Scheme 2.1 Synthesis of ionic liquid [bmim][PF6] 33 Scheme 2.2 Synthesis of polycap rolactone in ionic liquid [bmim][PF6] 36

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vList of Symbols ad Abbreviations 1H Isotope of hydrogen with mass of 1 amu 13C Isotope of Carbon with mass of 13 amu Ac Acetyl AK Lipase from Pseudomonas fluorescens AYS Lipase from Candida rugosa oC Temperature in degree Celsius CDCL3 Deuterated chloroform CH3OD Deuterated methanol COSY Correlation Spectroscopy DEPT Distortionless Enhancement by Polarization Transfer DEPT-135 Distortionless Enhancement by Polarization Transfer at a flip angle of 135 degree DMSO-6 Deuterated dimethyl sulfoxide h hour(hours) j Coupling constant MHz Megahertz MS Mass Spectrometry PCL Poly caprolactone PPL Porcine pancreatic lipase ROP Ring opening polymerization Rf Retention factor SL Sophorolipid

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vi CHEMO-ENZYMATIC ROUT E TO THE SYNTHESIS OF BIODEGRADABLE POLYMERS AND GLYCOLIPID ANALOGS Surbhi Bhatt ABSTRACT New catalytic synthetic methods in organic ch emistry that satisfy increasingly stringent environmental constraints are in great demand by the pharmaceutical and chemical industries. Studies over last 15 years have revealed that activity of enzymes can be increased in organic solvents rather than th eir natural aqueous environment. Because of their ease of use, high selectivity and envi ronment friendliness, enzymes are enjoying increasing popularity in t odays synthesis world. Chapter 1 describes chemo-enzymatic synthesi s of various glycolipid analogs. A highly regioselective macrolactonization was achieved using lipase from Candida antarctica as a catalyst. It also descri bes evaluation of lipases from different source and their efficiency in catalyzing the macrolactonization reaction. Th ese analogs were synthesized using commercially available agriculture based disaccharides (maltose, lactose, cellobiose, melibiose). These glycolipid an alogues have potential applications in the cosmetic industry, formulation, food production, and pharmaceutical industry. In Chapter 2, ring opening polymerization of -caprolactone in ioni c liquid, [bmim][PF6] was investigated. A comparative study of ROP in different solvents (t oluene, Ionic liquid, and bulk condition) was conducted. Effect of time and enzyme concentration on molecular weight and % yield was investigat ed. It was concluded that enzymatic ring opening polymerization of -caprolactone in ionic liquid, [bmim][PF6 ] is a very

PAGE 9

vii competitive and environmental friendly way of synthesizing high molecular weight polyesters.

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1 CHAPTER 1 Lipase-Catalyzed Synthesis of Glycolipid analogs 1.1 Introduction Glycolipids are a particularly importan t class of cell membrane components. Principally they are only found in the exte rior of the cell wall. Various bioactive structures possessing macrolidic structures or an aliphatic chain attached to a sugar moiety are abundant in nature and are interesting syntheti c targets due to challenges associated with the synthesi s of highly functionalized glyc olipid analogs from readily available synthons. Glycolipids are interest ing group of natural products that have complex structures and intere sting biological activities ( Figure 1.1 ). Sophorolipids (SLs) belong to such a class of glycolipids which contain a -hydroxy acid as an agylcon attached to a saccharide backbone ( Figure 1.1 ).

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2 O O O OH HO HO HO O O O O O O O O HO HO HO O O O O O O OH HO HO HO HO O O O HO HO O O H O O O HO HO O HO OH O HO O Tricolorin A Tricolorin G Sophorolipid LactoneO O HO O O HO O O H O O O HO O O O HO O O O O Simonin IO O O OH HO HO HO O O O O HO O O O O O OH O O O O O O Woodrospin IHO OH O O O HO HO HO HO OH O HO HO OH O Sophorolipid AcidO O OH O HO HO HO O O HO HO HO O OH N H O Globoside-3O O O O O OH OH HO HO OH OH O O O O G l uco l i ps i n Figure 1.1 Various natural Glycolipids

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3 Sophorolipids produced by the yeast Candida bombicola are amphiphilic molecules of growing commercial inte rest as biodegradable emulsifiers. Such molecules find applications in the petroleum, pharm aceuticals, and food processing industries where they can be used to reduce surface tens ion, stabilize emulsion, and promote foaming.1 Compared to their synthetic counterparts biosurfactents offer some distinct advantages: they are produced from renewable resources they are nontoxic a nd biodegradable, they are effective under extreme conditions in sm all quantities. Sophoro lipids consist of a sophorose molecule linked to a hydroxyl group at the penultimate position of, most often, a C-18 fatty acid.2 Native SL is rather complex mixt ure of up to 14 different compounds, both lactone and open chain acid form.3 1.2 Bioactivity of glycolipids Although the biological properties of glyco lipids have not been fully assessed, a closer look at this class of natural products seems highly promising. They are typically involved in intercellular rec ognition processes like cell a ggregation and dissociation and initiation of cell division. Globoside-3 ( Figure 1.1 ) is a minor component of tissues, deficiency of which l eads to Fabrys disease.4 Yet another glycolipid, galactose cereboside which has structure si milar to globoside with gala ctose as head group is an important component of membranes of nerve tissu e and is associated with disease such as multiple sclerosis.4 Tricolorin A ( Figure 1.1 ) is isolated from a plant called Ipomoea tricolor which acts as a natural herbicide. It has been reported to have significant cytotoxicity against cultured P-388 and human breast cancer cell lines.5-7 Woodrosin I,

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4 resin glycoside isolated from the stems of Ipomoea toberosa L seems highly promising in view of the existing data on the use of glyc olipids in general for the treatment of severe immune disorders.8 The existing data indicates the potential of sophorolipids as immunomodulators for Parkinson's disease, Alzheimer's disease, psoriasis, AIDS treatment, as well as for antiviral immunostimulation.9,10 Also there have been reports of SLs causing differentiation and protein kinase C i nhibition in the HL60 leukemia cell line.11 Glucolipsin, a macrolidic glycolipid produced by Streptomyces purpurogenisclerotics is known to act as a glukinase inhibitor.12 Simonin 1 has been isolated from plant Ipomoea batatas is used in Brazilian folk medicine.13 1.3 Traditional Syntheses of Glycolipids Whole cell biocatalytic approaches have been investigated for synthesis of microbial sophorolipids. For instance, Gro ss et. al. have used fermentation for the synthesis of Sophorolipids.14 In this strategy industrial waste containing fatty combined with glucose were evaluated both in batch and fed-batch processes for the production of sophorolipids using Candida bombicola ATCC 22214. Maximum sophorolipid yields of 120 g/L and productivity of 12.0 g/L per day was obtained by fed batch fermentation using tallow fatty acid residue. To improve the yields Feed of coconut fatty acid residues was used which resulted in increase of the cell production 14 ( Chart 1.1)

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5 Sophorolipidsglucidic and lipidic substrate i.e. glucose, mannose lactose and vegetable oil or animal fat Candida bombicola or Candida apicola Chart 1.1 Flow chart for fermentation process Daniel et al. have developed an im proved two batch cultivation process ( Chart 1.2 ) using deproteinized whey as feed.15 They used the yeast C. curvatus in the first and the yeast C. bombicola in second step. The two step strategy was important because C. bombicola was not able to consume lactose dire ctly from the substrate deproteinized whey. This made it essential to use C. curvatus in the first cultivation step.

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6 WheyCrossflow and sterile filteration C u l t i v a t i o n o f C r y p t o c o c c u s c u r v a t u s A T C C 2 0 5 0 9 Consumption of lactose as carbon source Biomass production Singlecell oil production(SCO) Cell disruption and sterilization C u l t i v a t i o n o f C a n d i d a b o m b i c o l a A T C C 2 2 2 1 4 Use of the cell debris and glucose for growth Consumption of SCO for prod uction of sophorolipids Chart 1.2. Flow chart of the coupled process including filtration

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7 It is reported that the composition of natural sophorolip id mixture can be altered by the selective-feeding of lipophilic substrates.16, 17 For example, changing sunflower to canola oil resulted in a large increase ( 50 to 73%) of the lactonic portion of SLs.18 Unsaturated C-18 fatty acids such as oleic ac id may be incorporated unchanged into SLs and result in dramatic change in the their compositions.19,20 However, it is clear that this approach is largely limited to composition cha nge or incorporation of select agylcons. Clearly, a different synthetic approach is ne eded but the intricate structure of these glycolipids poses many synthetic difficulties an d consequently very lit tle effort has been devoted to this area. In the synthesis of macrolidic glycolipid s, one of the major challenge resides in the regioselective formation of the macrolactone ring to synthesize lactonic analogs. The formation of the macrolactone thus far has been accomplished by Yamaguchi21, Corey22, or Mitsunobu23 macrolactonization conditions. More recently use of ring closing metathesis reaction has been explored for formation of the macrolides woodrosin I, sophorolipid lactone and Tricolorin A ( Scheme 1.1 ). 24, 25

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8O RO O OH O RO O OR RO O O RCM O RO O OH O RO O OR RO O O D-glucose, D-Fucose OH Scheme 1.1 Retro-synthesis of a Tricolorin A intermediate by Furstner et al.24 Heathcock et. al. have reporte d synthesis of tricolorin A by selective macrolactonization using Yonemitsu protocol26 ( Scheme 1.2 ). Although very useful, these synthetic approaches to the formation of the macrolides require strategically placed reactive groups necessitating a number of protection/deprot ection steps resulting in a long synthetic sequence.

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9 O RO O OH O RO O OR RO O O O O O OH O H O H O H O O O O O O O O OH O H O H O O O O O O O O O OH O H O H O OR O RO OR OH O RO O OR RO O C5H11 COOH Tricolorin-A Scheme 1.2 Retrosynthesis of Tricolor in-A by Hitchcock et. al.26 Our goal is to develop a chemoenzymatic strategy well suited to the synthesis of well defined macrolidic glyco lipid analogs, which would otherwise be unavailable or difficult to synthesize, for subsequent evaluati on of their properties and bioactivities. The main advantages of usi ng this strategy would be a) low cost starting materials. b) changing number and variety of carbohydr ate residue in the head group and using different aliphatic hydrophobic chai n will provide a ha ndle to different analogs having different physical properties.

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10 c) non-toxic reaction conditions and biodegradable products. d) strategies applied are in accordance with the princi ples of green chemistry. 1.4 Experimental Section 1.4.1 Chemicals and enzymes. All reagents were purchased from comme rcial sources and used as received. All solvents were purified and dried prior to use by known literature procedure. Porcine pancreatic lipase (PPL) Type II crude (activity = 61 units/mg protein) and Candida rugosa lipase(AYS) Type VII (act ivity = 4570 units/gm protein) were purchased from Sigma Chemical Co. The lipase PS-30 from Pseudomonas cepacia (20,000 units/g) was obtained from Amano Enzymes Co. Ltd. The carrier fixed lipase Novozyme 435 (10,000 units/g from Candida anatarctica fraction B) was a gift from Novo Nordisk Inc. 1.4.2 Column chromatogrpahy Column chromatographic separations were performed over silica gel 60 (Silicycle Inc.). In a typical separation, silica gel was us ed to pack a glass column (5cm X 50cm) in the eluent (ethyl acetate/hexane mixture). The compounds were dissolved in a minimal volume of eluent and loaded onto the top of the silica bed in the column. Different fractions were subsequently eluted and m onitored by thin-layer chromatography (TLC). Fractions containing the purified compounds we re pooled together, and the solvent was evaporated to give the pure compound.

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11 1.4.3 Nuclear magnetic resonance 1H-NMR and 13C-NMR spectra were recorded using Bruker ARX-250, INOVA 400, and INOVA 500 spectrometer. Chemical shifts in parts per million are reported downfield from 0.0 ppm using deuterated chloroform or deuterated DMSO with trimethylsilane (TMS) as the internal reference. Unambiguous assignments were derived from COSY and HMBC spectra. 1.4.4 General synthesis of oct aacetate of lactose, maltos e, melibiose and cellobiose (2) In a 250 ml round bottom flask equippe d with reflux condenser 10 g (0.0277 mol)of disaccharide (maltose, cellobiose, melibiose, or la ctose) 8 g (0.0975 mol) of sodium acetate and 50 ml (0.489 mol) of acetic anhydride were added. The reaction assembly was protected from atmospheric moisture by CaCl2 guard tube. The reaction mixture was refluxed for 4 h, cooled to room temperature and worked up by precipitating out by adding it dropwise over stirring ice th e product precipitated out immediately which was then filtered, washed with icewater, and dried overnight under pressure in a vacuum oven to yield octaacetate of s ugar as a white solid (yield 96 %) 1.4.5 Synthesis of methyl 15-hrdroxypentadecanoate A 250 ml round bottom flask was charged with 10 g of pentadecalactone, 150 ml dry methanol and 2 ml 0.022 N freshly prep ared sodium methoxide. Reaction mixture

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12 was refluxed for 4 h and then cooled down to room temperature and then neutralized using glacial acetic acid. Th e reaction mixture was concentr ated by rotoevaporation and poured over stirring crushed ice dropwise to yield the product. The white product was filtered, washed with ice-water and dried ove r night under pressure in a vacuum oven. (95% recovered Yield). 1.4.6 Synthesis of compounds 3a-d In 100 ml round bottom flask 2 g (0.0028 mole 1 equiv.) of acetylated sugar and 1.168g (0.0043 mol, 1.5 equiv.) was taken. To th is mixture 40 ml of freshly distilled dichloromethane was added to the flask us ing syringe. The reaction mixture was then cooled down to 0 oC by placing it in a ice-bath. Then to this cold stirring mixture, 2.03 ml (0.01 mol, 5 equiv.) of boron trifluoride ethera te was added drop wise After the addition was complete the reaction mixture was remove d from ice bath and brought back to room temperature. It was stirred for 7 h at r oom temperature. The reaction mixture was protected from moisture using rubber septum and nitrogen was flushed through the round bottom flask during transferring solvent and catalyst. After 7 h, the reaction mixture was diluted by adding 20 mL of dichloromethan e and then neutralized using saturated solution of sodium bicarbonate. The organic layer was washed 3 times with 20 mL deionized water, dried over sodium sulfate, and concentrated by ro toevaporation. The viscous liquid was then purified using co lumn chromatography using hexane : ethyl acetate (60:40) eluent system. Same fractions were collecte d and dried under pressure in vacuum oven to yield pure coupled product 3a-d 50% yield.

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13 1.4.7 Synthesis of Compounds 4a-d Herein we give general pr ocedure for synthesis of 4a-d In 100 ml round bottom flask 1 g of Compound 3 and 2 mL 0.022 N fr eshly prepared sodium methoxide in methanol were added. The reaction mixture was protected from moisture using rubber septum and nitrogen was flushed through th e round bottom flask dur ing transferring solvent and reagent. The reaction was stirre d overnight at room temperature and then neutralized using glacial acetic acid. Th e reaction mixture wa s concentrated by rotoevaporation and poured over stirring crus hed ice dropwise to yield the product. The white product was filtered, washed with icewater and dried over ni ght under pressure in a vacuum oven (92% recovered yield). 1.4.8 Screening of lipase and general procedure of lipase catalyzed mecrolactonization and lipase catalyzed ester hydrolysis In 50 ml round bottom flask appropriate substrate ( 4a-d ) was dried overnight in vacuum oven. Enzymes were dried over P2O5 overnight and enzymes were transferred to reaction flask in nitrogen bag to maintain st rictly dry conditions. To the reaction flask then freshly distilled THF was added using syringe. Reaction was stirred for 96 h at 30 oC. Novozyme 435 was found to be the only en zyme that catalyzed the lactonization reaction. The reaction mixture was then filte red through a bed of celite and concentrated by rotoevaporation. The re sulting crude product was purified by wet column chromatography using methanol a nd dichloromethane as eluent.

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141.5 Results and Discussion Lipase catalyzed acylations and transesterif ication reactions have been evaluated for formation of lactones. The hydrolytic enzymes lipases are known to catalyze macrolactone formation by intraesterification in absence of water or other nucleophiles such as alcohols.27 Stemming from our interest in bioc atalysis, we deve loped efficient short synthetic route to compounds 5a-d ( Figure 1.2) exploiting the regi oselectivity of the lipases ( Scheme 1.3) O O O CH2OH CH2OH OH OH OH OH O OH C H2C H2O O H 13 O O O CH2OH CH2OH OH OH OH OH O OH C H2C H2O O H 13 O O CH2OH OH OH O OH C H2OH O OH C H2C H2O O O O O CH2CH2OH OH OH OH OH OH C H2C H2O O O 13 13 5a 5b 5c 5d Figure 1.2 Different glycolipid analogs

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15 In a previous study by Bisht et. al. it was demonstrated that the sophorose, the glycon portion of sophorolipids, is able to f it in the active site of the lipase CA (from Candida antarctica ) and that the lipase CA catalyzed macrolactone formation between 17-hydroxyoctadec-9-enoic acid s ubunit and the sophorose backbone.28 Utilization of readily available disaccharides maltose, lactose, cellobiose and mellibiose ( Figure 1.3 ) is highly desired because of their origin in agri culture based feedstock and we have utilized them in synthesis of macro lidic glycolipids analogs. O O O CH2OH CH2OH OH OH OH OH OH OH O O O CH2OH CH2OH OH OH OH OH OH OH O O O CH2OH CH2OH OH OH OH OH OH OH O CH2OH OH OH OH O O OH C H2OH OH OH Lactose Cellobiose Maltose Melibiose Figure 1.3 Different disaccharide used as starting material The aglycon, 15-hydroxypentadecan oic acid was obtained from the -pentadecalactone, which is a well known substrate for the Candida antarctica lipase.29

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16 HO OH HO HO HO O OH O OH O O OH OH HO HO HO O OH O OH O O 12 OH OH HO HO HO OH OH O OH O O 12Maltose or Mellibiose HO Methyl 15-hydroxypentadecanoate OH HO Scheme 1.3 Synthetic approach to different glycolipid analogs. Ring opening reaction of pentadecalactone using sodium methoxide in methanol provided methyl 15-hydroxy pent adecanoate in 93% yield ( Scheme 1.4 ). O O HOOMe O MeOH reflux 5h 11 11 NaOMe 1 Scheme 1.4 Synthesis of 15-hydroxy pentadecanoate O O O HO OH HO OH HO OH HO OH O O O OAc OAc OAc O A c O A c OAc OAc OA c NaOAc (CH3CO)2O reflux, 5h 2a-d a = Maltose b = Melibiose c = Lactose d = Cellobiose Scheme 1.5 Acetylation of disaccharide

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17 All the disaccharides (maltose, lactose, melibiose and cellobiose) were peracetylated using sodium acetate and acetic anhydride to form respective octaacetates 2a-d in nearly quantitative yield ( Scheme 1.5 ). Taking advantage of the increased anomeric reactivity, 2a-d were directly glycosylated with met hyl 15hydroxypenatdecanoate in the presence of boron trifluoride etherate in freshly distilled anhydrous dichloromethane to afford 3a-d in around 50% yields, respectively ( Scheme 1.6 ). The stereochemical assignments were confirmed by 1H NMR, where the J12 value for H-1 (8 Hz) indicated the -Dconfiguration for the side chain. Gl obal deprotection of the peracetates 3a-d was achieved in nearly quantitative yield upon stirring with sodi um methoxide in anhydrous methanol for five hours at room temperature. Structure assignments of 4a-d were confirmed using extensive spectrometric analysis. The absence of the acetoxy methyl resonances in the proton spectra at ~ 1.962.19 ppm and in the carbon-13 NMR spectra at ~75 ppm confirmed the global deacetylation. Also, absent were acetoxy es ter carbonyl (C=O) resonances at ~170 ppm in the carbon-13 spectra. The resonance signa l of the methyl ester was observed at ~3.5 ppm and ~50 in the proton and car bon-13 NMR spectra, respectively.

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18O O O OAc OAc OAc OAc OAc OAc OAc OAc HOOMe O O O O OAc O OAc OAc OAc OAc OAc OAc O OMe BF3.OEt2, CH2Cl2O O O HO O HO OH HO OH HO OH O OMe + 10 h, rt. NaOMe, MeOH 5 h, rt. 11 10 10 2a-d 1 3a-d 4a-d a = maltose b= Melibiose c= lactose d= cellobiose Scheme 1.6 Synthesis of 4(a-d)

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19 1.5.1 Screening of Lipase The screening for lipases were carried out in 50 mL round botto m flasks using 1:1 substrate to lipase ratio (w/w). 13 O O O O H O O H OH O H OH O H O O O O O O H O O H OH O H OH O H OH O 10 OMe PPL THF 96 h PS-30 THF 96 h AK THF 96h AYS THF 96 h Novozyme-435, THF 96 h Scheme 1.7 Screening different lipases for the regioselective macrolactonization of substrate 4a These reactions were carried out in dry TH F using different lipases for 96 h. Strictly anhydrous conditions were maintained. Since the size of scissile fatty acid binding pocket in lipase is known to vary considerably30, five different enzymes were used namely, PS30(from Pseudomonas cepacia ), PPL (porcine pancreat ic lipase), AK, AYS (from Candida rugosa ) and Novozyme-435( immo bilized preparation of Candida antartica )

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20 ( Scheme 1.7 ). While no activity was seen for PPL, PS-30, AK and AYS in the tested reaction media, formation of a prominent product (different Rf values comapared to the substrate ) was observed within 96 h upon incubation of 15-( 4 O -D-glucopyranosyl-Dglucose)-pentadecanoate 4a with lipase from Candida antarctica ( Novozyme). With these observations further st udies were done using Novozyme-435. 1.5.2 Lipase-catalyzed macrolactonization The final step of our synthetic sche me was the regioselective formation of macrolactone resulting in compound 5a and 5b. In a previous study Bisht et. al have reported enzyme catalyzed formation of m acrolactone ring at C-6 position when sophorolipid methyl ester was taken as a substrate.28 With 4a and 4b in hand, the lipase catalyzed macrolactonization was attempted. The reactions were performed in anhydrous THF at 30oC for 96 hours. Control reactions were setup similarly but without added lipase. Of all the lipases tested only the lipase CAL led to lactonic analogs 5a and 5b ( Scheme 1.8 ) which had a Rf higher than the starting compound when compared on a thin layer chromatography in methanol: CH2Cl2 (1:3). Detail spectral analysis, described later in this chapter, was undertaken to establish the st ructure as the compounds as 5a and 5b Control reactions set similarly but without added lipase did not catalyze formation of the compounds 5a and 5b This observation clearly establ ishes the process being lipase catalyzed macrolactonization.

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21 O CH2OH OH OH OH O O OH C H2OH O OH OCH3O O CH2OH OH OH O OH C H2OH O OH C H2C H2O O O 13 O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 13 O O O CH2CH2OH OH OH OH OH OH C H2C H2O O O 94b 5b30 oC 96 h Novozyme-435/THF 94a30 oC 96 h Novozyme-435/THF5a Scheme 1.8 Lipase catalyzed macrolactonization. 1.5.31 Lipase catalyzed hydrolysis of the methyl ester Unlike the reaction of the lipase with compounds 4a and 4b in reaction set up with compounds 4c and 4d, the lipase catalyzed reaction with Novozym 435( Scheme 1.9) lead to a product with lower Rf in a thin layer chroma tography experiment in methanol: CH2Cl2 (1:3).

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22O O O CH2OH CH2OH OH OH OH OH O OH OCH3O O O O CH2OH CH2OH OH OH OH OH O OH OCH3O O O O CH2OH CH2OH OH OH OH OH O OH OH O O O O CH2OH CH2OH OH OH OH OH O OH OH O 9 9 4c 4d 5c 5d 30 oC 96 h Novozyme-435/THF 30 oC 96 h Novozyme-435/THF 9 9 Scheme 1.9 Lipase catalyzed hydrolysis of methyl ester The 1H NMR spectrum of the products 5c and 5d did not have the resonance for methyl ester group which shows up at ~3.5 ppm in the precursors but a ne w resonance at ~12 ppm was present, possibly for a free carboxylic acid group. The 13C-NMR experiment also did not contain a resonance for the ester methyl (~ 50 ppm ) and the carbonyl resonance was present at ~168 ppm Th e HMBC experiment, to probe long range 13C-1H couplings revealed that the carbonyl carbon (C -1) of the aliphatic chain did not show coupling with any of the hydrogens of the disaccharide head group. In the DEPT -135 NMR spectrum of compound 5(c,d) resonance for methoxy methyl group was missing but there was no movement in C6 resonance or any other carbon when compared with the starting material( Figure 1.4) .These facts suggested the hydrolysis of the methyl ester of the aliphatic chain yielding acidic analog 5(c,d). Which was confirmed by the mass spectral analysis of the compounds 5c ( ESIMS m/z 582 (M+H) and 5d (ESIMS m/z 582 (M+H); calculated M+ 582).

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23 Figure 1.4 DEPT showing lipase catalyzed hydr olysis of methyl ester 1.5.4 Structure elucidation of ma crolactone lipids 5a and 5b Extensive structural analysis of produc ts isolated from the lipase catalyzed reactions was undertaken using 1H, 13C and 2D-NMR spectra. Detailed NMR analysis was carried out on similar glycolipid like structures by Bisht et al. 28 and noted limited utility of the 1H NMR spectra in establishing the st ructure and assignments of various signals. 13C NMR spectrum because of its wider ra nge (0-200 ppm) was better suited for assignment of different carbons in these comp lex glycolipid analogs structures. COSY, 13C-NMR, DEPT and HMBC ( carbonproton l ong range correlation) were utilized in assignments of different resonances.

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24 The spectra of the compounds 5a and 5b were compared to their corresponding starting materials, 4a and 4b In the 1H NMR spectra the reso nance for methyl ester protons (~3.5 ppm) was not present in the products 5a and 5b and also a resonance for the free carboxylic acid (~ 12 ppm) was not observed, suggesting the formation of macrolactone. Though the proton resonances of the sugar skeleton appeared perturbed it was not no possible to decipher any useful observation due to overlapping of the various resonances. The carbon 13 NMR has been previously used by Bisht et al28 and we decided to utilize it in our case. The car bon spectra was edited using a DEPT pulse sequence and the DEPT-135 of products were compared with starting materials .The wide distribution of the resonance freque ncies in the DEPT spectra allowed for assignment of each carbon resonance using COSY, HETCOR and HMBC correlations Comparison of the DEPT spectra of the pr oduct and the respective starting compound allowed for interesting observation to be made ( Figure 1.5) For example, in DEPT for compound 5a signal for methyl ester group was missing and downfield shift of 4 ppm was observed for C-6 carbon The HMBC sp ectra also showed three bond coupling between the carbonyl carbon (C1, in the side chain) and C-6" hydrogens, confirming the formation of macrolactone between the carbonyl carbon of the side chain and C-6 of the maltose head group ( Figure 1.6 ). These observations unequivocally established the formation of lactone ring at C-6 position and the structure of the compounds were established as 5a and 5b

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25 Figure 1.5 DEPT-135 showing lipase catalyzed m acrolactonization at C6 position.

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26 Figure 1.6 HMBC of product 5a showing correlation between C-6 and C-1 1.6 Conclusion In summary we have achieved highly re gioselective formati on of macrolactone catalyzed by lipase. The selectivity demonstr ated by the lipase is unparalleled in organic synthesis and without involvi ng extensive protection/deprot ection chemistries. These sophorolipid-type glyco lipid analogues have potential applications in the cosmetic industry, formulation, food production, and even utility in technical purpose such as oil pollution abatement of sea water can be envisaged as these biosurfactants can enhance the emulsification of hydrocarbons increasing th eir availability for microbial degradation. These analogs will be evaluated for their biological and surface properties

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271.7 References (1) Haferburg, D.; Hommel, R.; Claus, R.; Kleber, H.P. Adv. Biochem. Eng. Biotech. 1986 33 53. (2) Gobbert, U.; Lang, S.; Wagner, F. Biotechnol. Lett. 1986, 6 225. (3) Davila, A. M.; Marchal, R.; Monin, M.; Vandecasteele, J.P. J. Chromatogr. 1993 648, 139 (4) Dmoulin, F.; Lafont, D.; Boullagne r, P.; Mackenzie, G.; Mehi, G. H.; Goodby, J.W. J. Am. Chem. Soc. 2002 124, 13737. (5) Perada-Miranda, R.; Mata, R.; Anay a, A.L.; Wickramaratne, M.; Pezzuto, J.M.; Kinghorn, A.D.; J. Nat. Prod. 1993 56, 571. (6) Bah, M.; Perada-Miranda, R. Tetrahedron 1996 52 13063. (7) Bah, M.; Perada-Miranda, R. Tetrahedron, 1997 53 9007. (8) Ono M., Nakagama K., Kawasaki T., Miyarama, Chem. Pharm. Bull ., 1993 41 1925 (9) Piljac, G.; Piljac, V. US Patent 5,514,661, 1996 (10) Piljac, G.; Piljac, V. US Patent 5,455,232, 1995 (11) Isoda, H.; Kitamoto, D.; Sh inmoto, H.; Matsumura, M.; Nakahara, T; Biosci. Biotechnol. Biochem. 1997 61, 609. (12) Furstner, A.; Radkowski, K ., Grabowski, J.; Wirtz, C.; Mynott, R. J. Org. Chem. 2000 65 8758. (13) Noda, N.; Yoda, S.; Kawasaki, T.; Miyahara, I. Chem. Pharm. Bull, 1992 40, 3163. (14) Shah, V.; Arthur, F. P.; Doncel, G. F.; Gross, R. US Pat. 2004

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28 (15) Daniel, H.J.; Otto, R.T.; Binder, M.; Reuss, M.; Syldatk, C. Appl. Microbiol. Biotechnol. 1999 51 40. (16) Gorin, P. A. J.; Spencer, J. F. T.; Tulloch, A. P. Can. J. Chem 1961 39 846. (17) Tulloch, A. P.; Spencer, J. F. T.; Gorin, P. A. J. Can.J. Chem 1962 40 1326. (18) Zhou, Q. H.; Kosaric, N J. Am. Oil Chem. Soc. 1995 72, 67. (19) Asmer, H.J.; Lang, S.; Wagner, F.; Vray, V. J. Am. Oil Chem. Soc. 1988 65, 1460. (20) Rau, U.; Manzke, C.; Wagner, F. Biotechnol. Lett. 1996 18 149. (21) Larson, D. P.; Heathcock, C. H.; J. Org. Chem. 1997 62 8406. (22) Carrillo, R.; Martin, V.S.; Martin, T. Tetrahedron Letters, 2004 45, 5215. (23) Selle`s, P.; Lett, R. Tetrahedron Letters, 2002 43, 4627. (24) Furstner, A. Muller, T. J. Am. Chem. Soc. 1999 121, 7814. (25) Furstner, A.; Radkowski, K ., Grabowski, J.; Wirtz, C.; Mynott, R. J. Org. Chem. 2000, 65 8758. (26) Hikota, M.; Sakurai, Y.; Horita, K.; Yonemitsu, O. Tetrahedron Lett. 1990, 31 6367. (27) Gargouri, M.; Drouet, P.; Legoy, D. M. Journal of Biotechnology 2002 92 259. (28) Bisht, K.S.; Gross R.A.; Kaplan L. David j.Org.Chem 1998 64, 780. (29) Bisht, K.S.; Henderson, L.A.; Gross, R.A.; Kaplan, D.L.; Swift, G. Macromolecules 1997 30, 2705. (30) Faber, K. 4th Eds. Biotransformations In Or ganic Chemistry. Springer-Verlag Berlin Heidelberg, 2000, 13.

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29 CHAPTER 2 Ring opening polymerization of -Caprolactone in ionic liquid 2.1 Introduction Today, polymers find applications not only in commodities or structural items but also in biomaterials, i.e. implants, artifici al limbs, drug delivery, etc. Polymers have nearly replaced traditional materials such as wood, steel, aluminum, and glass in many applications because of rather simpler proce ss of polymerization and the vast variety of polymers that can be synthesized with en tirely different properties. Degradable polyesters, for their biocompatib ility, have wide range of uses in medical applications and other environmental app lications. The ester linkage is su sceptible to hydrolysis and has been shown to degrade both hydr olytically and enzymaticall y. The degradation products of the biodegradable polyesters are typically hydroxy acids, such as glycolic acid and lactic acid that are generally recognized as being non-toxic. For these reasons there has been a large volume of research into the biod egradable polyester family in the past few decades. One of the most widely used monomer in synthesis of polyesters is caprolactone. Poly ( -caprolactone), for its biodegradability and permeability into various molecules1 can be used in drug delivery systems. PCL can also be potentially used as herbicide encapsulator for slow de livery of herbicide over a period of time.2 Biocompatibility, slow degrada tion and higher strength makes polycaprolactone fibers especially suitable for tissue engineering.3

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30 Although polymers are virtue of the m odern world, the environmental pollution caused by the methodologies used has raised increasing concerns. The increasing need for sustainable growth and development make s it imperative to find ways to limit production of unwanted side-products, to lo wer energy requirements and costs, and to design and introduce biodegrada ble polymers where environmen tal disposal is required. There has been an exponential increase in in terest in area of in vitro enzyme catalyzed organic reactions, since many families of en zymes can be utilized for transformation of not only their natural substr ates but a wide range of unnatural compounds, yielding a variety of useful materials.4, 5 Isolated enzymes are being used as off-the-shelf catalysts because of the ease of handling and product is olation. Enzymes offer promising substrate conversion, high selectivity (enantioand regioselec tivity), recyclability, and biocompatibility (non-toxic catalysts). In addition to the aforementioned remarkable features, enzymes are part of a sustainable en vironment. They come from natural systems and when they are degraded, their constituent amino acids are recycled back into natural substances, which makes them particularly attractive to replace potentially toxic heavy metal catalysts for polymer synthesis. All naturally occurring polymers are produ ced in vivo by enzymatic catalysis. In last few years, in vitro synthesis of pol ymers through enzymatic catalysis (enzymatic polymerization) has been extensively developed.6 Besides being very selective, enzymes are dynamic and sometimes very generous in recognizing variety of substrates in vitro This property of enzymes allows them to catal yze the synthesis of not only some natural polymers but variety of unnatural polymers t oo. Thus, the target macromolecules for

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31 enzymatic polymerization have been polysaccharides7, polyesters, polycarbonates, poly(amino acids)8 vinyl polymers, etc. Enzymatic polymerization has been receiving increasing attention as a new environmentally friendly method of polymer s ynthesis, in contrast to the chemical methods, which generally need harsh conditions and metallic catalysts that must be completely removed especially for medical applications. Furthermore, enzymatic polymerization can offer a novel method to produce polymers that are difficult to be synthesized by conven tional polymerization.9 Among the various polymerization methods, ring-opening polymerization is an important alternative route because leaving groups that can limit monomer conversion or degree of pol ymerization are not generated during polymerization.10 Enzymatic ring-opening polymer ization of lactones has been investigated, for lactone of small-size (4-membered)11, medium-size (6and 7membered) lactones 12-14, and large size (12-, 13-, 15-, 16-membered).15-18 It has been observed that enzymatic ring opening of the macrolactones yield much higher rate of polymerization and molecular weight of the polyester formed compared to the polymerization catalyzed by traditional chemical catalysts.19 In spite of all the benefits of enzymatic catalysis their utility in polymerization reaction is greatly limited due insolubility of the monomer and the growing polymer chain in the non polar solven ts, such as toluene and he xanes, which are typically employed in such reactions. Although, enzyma tic catalysis has been reported in polar nonprotic solvents, e.g., DMF, DMSO, pyridine, etc. but with significant loss of enzyme activity.20, 21 To avoid inactivation of the enzyme in polar solvents, polymerizations reaction especially of liquid or low melting monomers have been carried out without

PAGE 41

32 added solvent, i.e., in bulk. However, ever in creasing viscosity of the reaction mixture, as a result of the growing polymer chain, has been held responsible for low molecular weight of the resulting polymer.22 There clearly exists a need to evaluate other solvent systems for enzymatic polymerization reaction. In recent years, room temperature ionic liquids have received increasing attention as green solvents for wi de range of reactions.23 Room temperature ionic liquids are organic salts whose ions do not pack well and remain li quid at room temperature.24 Ionic liquids provide advantages w ith respect to catalyst recove ry, product separation, low toxicity, non-flammability, a nd re-usability. Ionic liquids are able to dissolve a wide variety of relatively insoluble organic and inorganic compounds to very high concentrations. Because of th eir above stated properties i onic liquids (ILs) have been evaluated as alternative solvents for lipase catalyzed reactions.25-27 They have also been widely employed for synthetic organic reactions.28-30 It has been acknowledged that ionic liquids can be engine ered to make them process-compatible by selecting appropriate cation and anion.31, 32 Although a variety of ionic liqui ds have been synthesized and studied; ionic liquid field cont inues to be extensively dominated by imidazolium salts with fluorine containing anions ( Scheme 2.1).

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33N N Br CH2Cl250h, refluxN N BrBu HPF6N N Bu PF6 -85% 90% 1 Scheme 2.1 Synthesis of ionic liquid [bmim][PF6] An important research focus of our labor atories continues to be the syntheses of biodegradable polymers through environmen t friendly methodologies. The enzymatic ring opening polymerization of caprolactone ( Scheme 2.2) in ionic liquid [bmim][PF6] 1 ( Scheme 2.1) is an approach to efficiently synthesize high molecular weight biodegradable polymers using bio-friendly procedures, which is yet another step towards green chemistry. Ionic liquids have polarities similar to pol ar organic solvents yet they do not inactivate lipases. In fact the polarit y of the ILs is expected to enhance the solubility of the monomers a nd the oligomers, and thus facilitate the enzymatic reaction and lead to possibilities of higher molecular weights. Several lipases are known to have enhanced catalytic activity in 1-butyl-3methylimidazolium hexafluorophosphate ionic liquid.33 This prompted us to attempt enzy me-catalyzed polyester synthesis in [bmim][PF6].

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342.2 Experimental Section Lipase Novozyme-435 was kindly provided by Novo Nordisk Bioindustrial, Inc. caprolactone monomer was bought from Sigm a Aldrich and was distilled before use. 2.2.1 Procedure for polymerization. The monomer and lipase were dr ied (in drying pistol over P2O5, at 50 C/0.1 mm of Hg; 15 h) separately in 6 mL reaction vials. In a glove bag maintained under nitrogen atmosphere, enzymes were transferred to th e vials containing monomers and vials were capped using rubber septa. Then ionic liqui d was added using 500 L syringe and vials were placed in constant temperature oil bath maintained at 60 C for predetermined time. Reactions were terminated by adding cold chlo roform to the reaction vial and removal of enzyme by filtration (glass frit ted filter, medium pore porosit y). The filtered enzyme was washed with chloroform and the filtrates were combined. Polymers were precipitated by adding chloroform solution drop wise to stirri ng methanol placed in ice bath. Polymers were separated by vacuum filtration using Hirch funnel and Millipore filter paper (0.45 m). The polymers were dried under vacuum to constant weight and dissolved in chloroform (HPLC grade) and chloroform-d for GPC analyses and NMR spectral data, respectively. 2.2.2 Preparation of 1-butyl-3-methyl imidazolium hexaflourophosphate: A) 1-Butyl-3-methylimidazolium bromide. A 250 ml, three-necked, round-bottom flask was equipped with a heat ing oil bath, a nitrogen in let adapter, an internal thermometer adapter, and a reflux condenser The flask was flushed with nitrogen and charged with 50 ml (0.06 mol) of freshl y distilled N-methylimidazole, 50 mL of

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35 acetonitrile and 67 mL(0.06 mol) of 1-bromobutane, and brought to gentle reflux (75-80 C internal temperature). The solution was heat ed under reflux for 48 h and then cooled to room temperature. The volatile material was removed from resulting yellow solution under reduced pressure. The remaining light yellow oil was re-dissolved in dry acetonitrile (50 mL) and added dropwise to well stirred 200 mL dry ethyl acetate. The imidazolium salt began to crystallize almost immediately. After the addition was complete the flask was put in an ice bath and cooled down to 0 oC for 2h. the supernated solution was removed via filtration and the re sulting white solid wa s dried under reduced pressure for 6 h to yield butyl-3-met hylimidazolium bromide in 85 % yield. B) 1Butyl-3-methylimidazolium hexafluorophosphate. A 200 mL, one-necked, round bottom flask was charged with 10 g (0.046 mol, 1 equiv) of 1-butyl-3methylimidazolium bromide and 8.9 g (0.0506 mol, 1.1 equiv) of sodium hexafluorophosphate in 16 mL distilled water. The reaction mixture was stirred at room temperature for 2 hr affording a two-phase sy stem. The organic phase was washed with 3 X 10 mL of water and dried under reduced pres sure. Then 20 mL of dichloromethane and anhydrous sodium sulfate were added. After 1 hr the suspension was filtered and volatile material was removed under reduced pressure The resulting ionic liquid a light yellow viscous liquid (80% yield) was the dried under vacuum for 6 hr. 2.2.3 Molecular weight Measurements Molecular weights were measured by gel permeation chromatography (GPC) using a Shimadzu HPLC system equipped with a model LC-10ADvp pump, model SIL 10A auto injector, model RID 10A refractiv e index detector (R I), model SPD-10AV UVvis detector, and waters HR 4E styragel column. Chloroform (HPLC grade) was used as

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36 eluent at a flow rate of 1.0 ml/min. The sa mple concentration and injection volumes were 0.5% (w/v) and 50 L, respectively. EzChrome Elite, Scientific Software Inc., was used to calculate molecular weights based on a calibration curve generated by narrow molecular weight distribution polystyrene standards. 2.3 Results and Discussion O OH O 4 O O Novozyme-435 [BMIM]PF6 Scheme 2.2 Synthesis of polycaprolactone in ionic liquid [bmim][PF6] Poly caprolactone (PCL) ( Scheme2.2 ) is one of the most inve stigated environmentally biodegradable synthetic polymers due to fac ile accessibility, variable biodegradability and good mechanical properties.34 It has been shown in our lab previously that Candida antarctica lipase (Lipase CA; Novozyme-435) exhibits promising result with respect to conversion, molecular weight and substrate selectivity in ring opening polymerization.35 Lipase CA is known to have high cataly tic activity for tran sesterification in [bmim][PF6. 36, 37 We investigated lipase cataly zed ring opening polymerization of caprolactone in ionic liquid, [bmim][PF6]. Different reaction conditions were studie d. For example to inve stigate the effect of the quantity of enzyme used, polymeri zation was done using different monomer to enzyme ratio. (Table 2.1 and Table 2.2). It was noticed that with increasing amount of

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37 the enzyme the yield was slightly lower than compared to when less enzyme was used and reaction was run for 40h. Also, the molecula r weight of the polymer was higher when larger amount of the lipase was used ( Table 2.2 ). To evaluate the performance of enzymes in catalyzing ring opening polym erization in ionic liquid ([bmim][PF6], polymerization in toluene and in absence of any added solvent was also investigated. Result of the comparative study in tolu ene, bulk and ionic liquid ([bmim][PF6] are included in Tables 2.1 and 2.2. It was observed that % yield was slightly higher in ionic liquid than other reaction medium studied (i.e. bulk and toluen e) (entries 1 and 2 in Table 2.1 and table 2.2). Polymerization in bulk resulte d in lower conversion (entries 3 and 4 in Table 2.1; entry 3 Table 2.2) partially because of the increased viscosity of the reaction medium. Higher yield in ionic liquid in co mparison to polymerization in toluene could be attributed to the polar nature of ionic liquid enhancing the solubility of growing polymer chains. Toluene being less polar so lubilizes the polymer chains in moderation resulting in slightly lower conversions. The results of reactions carried out at 75 oC for 40 h are summarized in Table 2.1 and Table 2.2.

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38Table 2.1 Comparison of polymerization in different mediums with monomer:enzyme:solvent ratio being 2:1:2 by weight at 75 oC for 40 h.. SN Monomer Vol.(mon.) Solvent Volume Novozyme(g) Yield(%) Mn Mw/Mn 1 -CL 200 L [bmim][PF6] 200 L 0.104 79 11884 2.3 2 -CL 200 L [bmim][PF6] 200 L 0.104 81 11548 2.2 3 -CL 200 L Bulk -------0.104 48 14812 2.5 4 -CL 200 L Bulk --------0.104 64 13119 2.3 5 -CL 200 L Toluene 200 L 0.104 75 12749 2.5 6 -CL 200 L Toluene 200 L 0.104 70 13285 2.5 Table 2.2 Comparison of polymerization in different mediums with monomer:enzyme:solvent ratio being 4:1:4 by weight at 75 oC. SN Monomer Vol.(Mon.) Solvent Volume Novozyme(g) Yield(%) Mn Mw/Mn 1 -CL 200 L [bmim][PF6] 200 L 0.051 60 13219 2.2 2 -CL 200 L [bmim][PF6] 200 L 0.051 45 12374 2.2 3 -CL 200 L Bulk -------0.051 40 16793 2.6 4 -CL 200 L Toluene 200 L 0.051 45 14384 2.5 5 -CL 200 L Toluene 200 L 0.051 50 14678 2.7 The lower yield in table 2.1 and tabl e 2.2 polymerization reactions run at 75 oC were somewhat disappointing. The lower cat alytic activity of the lipase, due to denaturation of enzymes at this high temperature could be the culprit and in fact survey of the literature revealed that CA lipase is known to show lower activity at higher temperature.38, 39 Hence these results of higher temperature on yields instigated us to run

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39 the polymerization reactions at lower temper atures and subsequently studies with the lipase were conducted at 60 oC. The reaction time was another important influencing factor and to study the progress of the polymerization reaction, the monomer conversion and the polymer molecular weight of the polymer were monitored with time ( Table 2.3 ). A time related study on polymerization of -CL in ionic liquid wa s carried out at 60 oC Table 2.3 Time-related study of polymerization in ionic liquid at 65 oC. SN Monomer AmountSolvent Time yield (%) MWn Mw/ Mn 1) Caprolactone 200 L [bmim][PF6]40min 41.2% 9615 1.9 2) Caprolactone 200 L [bmim][PF6]1.30h 62.6% 11225 2.2 3) Caprolactone 200 L [bmim][PF6]1.30h 58.35% 12576 2.0 4) Caprolactone 200 L [bmim][PF6]2.30h 61.95% 13308 2.0 5) Caprolactone 200 L [bmim][PF6]2.30h 65.40% 13286 2.0 6) Caprolactone 200 L [bmim][PF6]4.5h 77.15% 11018 2.0 7) Caprolactone 200 L [bmim][PF6]4.5h 81.65% 11018 2.0 8) Caprolactone 200 L [bmim][PF6]25h 78.9% 11300 2.0 9) Caprolactone 200 L [bmim][PF6]25h 77.95% 11171 2.0 10) Caprolactone 200 L [bmim][PF6]96h 91.50% 10614 1.9 11) Caprolactone 200 L [bmim][PF6]96h 91.25% 11191 2.0 It was observed that the monomer conversion increased with increasing reaction time and a linear relationship between reaction time and conversion was established ( Chart 2.2 ).

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40 It is important to point out that in polymerization reactions without added solvent the monomer conversion incr eases with time reaching a certain maximum (~90%) and does not increase with further increase in the re action time. It has been attributed to the fact that molecular weight of the polymer in creases with increasing conversion leading to higher viscosity, which makes the monomer inac cessible to the active site of the enzyme. In the ionic liquid, however, no su ch viscosity limitation exists and the linear increase in conversion with time is observed ( Chart 2.2 ). Molecular weight of the polycaprolactone formed increased with increasing conve rsion and reaction time and was 9615 g/mol within 40 minutes at 41.2 % conversion (entry 1 in Table 2.3). The molecular weight increased further to ~13300 g/mo l after 2.3 h at 63 % conversion ( Chart 2.1 ). However, the molecular weight did not show a continuous increase beyond 13300 g/mol even with increasing conversion to 90 %. In fact the molecular weight decreased somewhat to with progress of the polymer reaction leading to molecular weight of ~11000 g. mol at 91 % conversion ( Chart 2.1 ). Decrease in the molecular weight with the progress of the reaction beyond 63 % conversion is somewhat intriguing but not unexpected. Work in our laboratories and others have made simila r observation in reacti on carried out in bulk and in solvents40. It is believed at beyond a certain conversion the monom er availability to the enzyme decreases where as its accessibil ity to the polymer chain increases. With increasing accessibility to the growing polymer chain the enzyme catalyzed transesterification become mo re prevalent than polymer forming reaction leading to decrease in molecular weight.

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41 9615 11900 13297 11018 11235.5 10902 8000 9000 10000 11000 12000 13000 14000 0.41.32.34.52596 Time(h)Molecular weight Chart 2.1 Plot of molecular weight as function of time.

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42 41.5 60.47 63.67 79.4 78.22 91.5 30 40 50 60 70 80 90 100 0.41.32.34.52596Time (h)% Yield Chart 2.2 Plot of % yield as a function of time. The ionic liquids clearly are suited for enzymatic polymerization providing good polymer yield and easy enzyme recovery. The solv ent recovery was easy and quantitative. The reaction catalyzed in IL lead to higher polymer yield compar ed to the reaction performed in bulk conditions. The are similar to observation made in solvents such as toluene and are in agreement with previous reports.14,34 In essence it was observed that polymerization in ionic liquid [bmim][PF6] was competitive with the polymerization in toluene with better bicompatibilty and easier solvent recovery.

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43 2.4 Conclusion Novozyme-435 catalyzed polymerization of caprolactone in ionic liquid was found to be highly efficient for polyester pr oduction in an environm ent friendly strategy. By using this system a high % conversion a nd higher molecular weight polyesters were synthesized. It was concluded from above study that using lower equivalents (4:1, monomer : enzyme) of enzyme resulted in sl ightly higher molecular weight but with lower yields. Reaction time and higher temper ature does not affect enzyme catalysis in ionic liquid as much as it does in organic solvent under same conditions. With their vast array of applicati ons, synthesis of high molecular weight, biodegradable polyesters in environment frie ndly medium is an absolute necessity and ionic liquids serve as one of the best reaction medium for effective enzyme catalysis and production of high molecu lar weight polymers.

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47 Appendix 1

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48 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 9

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49 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 9

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50 Appendix 1 (Contd.) 13 O O O CH2CH2OH OH OH OH OH OH C H2C H2O O O

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51 Appendix 1 (Contd) 13 O O O CH2CH2OH OH OH OH OH OH C H2C H2O O O

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52 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 9

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53 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 9

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54 Appendix 1(Contd.)

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55 Appendix 1 (Contd.)

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56 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH OCH3O 9

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57 Appendix 1 (Contd.) O O O CH2OH CH2OH OH OH OH OH O OH C H2C H2O O H 13

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58 Appendix 1 (Contd.) O CH2OH OH OH OH O O OH C H2OH O OH OCH3O 9

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59 Appendix 1 (Contd.) O CH2OH OH OH O OH C H2OH O OH C H2C H2O O O 13

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60 Appendix 1 (Contd.) O CH2OH OH OH O OH C H2OH O OH C H2C H2O O O 13