2018 Divya Singh All Rights Reserved

2018

DIVYA SINGH

SYNTHESIS OF CADDISFLY INSPIRED POLYESTER ADHESIVE

A Thesis or Dissertation Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment of the Requirements for the Degree Master of Polymer Science

Divya Singh

May, 2018

Synthesis of Caddisfly Inspired Polyester Adhesive

Divya Singh

Thesis

Approved: Accepted:

Advisor Dean of the College Dr. Abraham Joy Dr. Eric Amis

Faculty Reader Interim Dean of the Graduate School Dr. Toshikazu Miyoshi Dr. Chand Midha

Department Chair Date Dr. Coleen Pugh

ABSTRACT

Adhesives have benefited the advancements in various fields of science and technology.

But when introduced to wet conditions, most synthetic adhesives fail to work efficiently1.

However, underwater organisms such mussels, caddisflies and bacteria can stick on to various underwater surfaces. Caddisfly larvae spins adhesive silk to assemble stationery structure consisting of leaves and twigs to construct protective shield around it in the presence of water2. Interestingly, these adhesive silk shows the presence of phosphorylated serine that is shown to play crucial roles in its adhesion underwater. The phosphate group is known for its ability to provide excellent coating stability through various interfacial interactions. In the case of mussels, a series of polyphenolic adhesive proteins are secreted from its foot to achieve underwater adhesion3. These phenolic groups have versatile crosslinking chemistry which can provide cohesive interactions to the adhesive. Herein, inspired from caddisfly adhesive silk and mussel foot proteins, we synthesized adhesive polymers which has phosphorylated serine and catechol mimetic molecules.

III

ACKNOWLEDGEMENT

I would like to acknowledge my advisor Dr. Abraham Joy for his constant support and motivation. I would also like to thank my reader Dr. Toshikazu Miyoshi for his time and suggestions. Also I would like to thank my mentor Amal Narayanan for teaching me the techniques and for his constant guidance.

IV TABLE OF CONTENTS

List of Figures

List of Schemes

CHAPTER I. Introduction ……………………………………………………………………………..9

1.1. Adhesion………………………………………………………………………….11

1.1.1. Mechanism of adhesion…………………………………………………….11

1.1.2. Cohesion……………………………………………………………………14

1.1.3. Failure modes of adhesion………………………………………………….14

1.2 Lap Shear test……………………………………………………………………..15

1.3. Adhesive technologies inspired by nature……….………………………………..18

1.3.1. Sandcastle worm…………………………………………………………..18

1.3.2. Mussels……………………………………………………………………19

1.3.3 Caddisfly……………………………………………………………………20

1.4. Role of Phosphorous in adhesion………………………………………………….22

II. Synthesis and characterization of N-substituted Diols ...... 23

2.1 Introduction ...... 23

2.2 Instrumentation ...... 24

2.3 Materials ...... …. 24

2.4 Synthesis of Ser mimic monomer ...... 25

2.5 Synthesis of cat mimic monomer ...... 26

2.6 Synthesis of soy mimic monomer...... 26

III. NMR Results and discussion of N-substituted Diol mimic Monomer ...... 28

3.1 Introduction ...... 28

3.2 Instrumentation...... 28

3.3 NMR data analyses ...... 29

IV. Synthesis of polyester derived from N-substituted diols...... 31

4.1 Introduction………………………………………………………………………….31

4.2 Materials ...... 31

4.3 Synthesis of parent polymer ...... 31

4.4 Deprotection of TBDMS ...... 32

4.5 Phosphorylation ...... 33

4.6 Deprotection of ethy ether group ...... 33

4.7 Deprotection of catechol ...... 34

V. Characterization and adhesive strength measurement ...... 35

5.1 Introduction ...... 35

5.2 Lap shear measurement ...... 35

5.3 Results and discussion...... 35

5.4 NMR data ...... 36

5.5 Lap shear data ...... 42

VI. Conclusion

REFERENCES

List of Figures

Figure Page

1.1 Adhesive bond energy vs type of bonds ...... 12

1.2 Adhesive and cohesive forces acting in adhesion; adhesive and cohesive failure ...... 14

1.3 a) Lap shear specimen; b) a tensile testing machine used for lap shear test ...... 16

1.4 DOPA mimic using free radical copolymerization of acrylamide ...... 18

1.5 synthesis of catechol with hydrophobic and electrostatic functional groups ...... 20

3.1 1H NMR of catechol mimic monomer ...... 28

3.2 1H NMR of soy mimic monomer ...... 29

3.3 1H NMR of serine mimic monomer ...... 29

5.1 1H NNMR spectra for the parent polymer ...... 36

5.2 1H NNMR spectra for the TBDMS deprotected polymer ...... 37

5.3 : 1H NNMR spectra for the phosphorylated polymer ...... 38

5.4 1H NNMR spectra for the deprotected ethyl ether group ...... 39

5.5 1H NNMR spectra for the deprotected catechol group ...... 40

5.6 Lap Shear results of the synthesized polymer ...... 41

List of Schemes Scheme Page

1. Synthesis scheme for serine mimic monomer 24

2. Synthesis of catechol mimic monomer 25

3. Synthesis of soy mimic monomer 26

4. Synthesis route from the parent polymer to the polyester adhesive 31

8 CHAPTER I

1. Introduction

Even though the adhesion technology has benefited various fields of science and technology, the one area which still is needs to benefit from it is the medical field. The tools usually used for repairing fractured or torn tissues include pins, screws, staples and pins. Metal plates and other metal devices have long been a part of dentistry. These routes of healing are conventional but have various disadvantages that include damage to the tissues surrounding them, possibility of bone regeneration that might lead to the need of them being replaced, poor dwelling to the bone. Thus, there is a need to replace these techniques with a more reliable adhesive technology that would also help in underwater surgeries. Developing an adhesive technology can be more or less divided into two methods – one being the synthetic route and the other inspired from nature. Synthetic adhesives include PMMA

(Polymethacrylates) and related polymers, polyurethanes and cyanoacrylates provides good mechanical but are not biodegradable or biocompatible to the desired level. There has been an extensive amount of research being conducted on Phosphorous containing monomers and polymers. Their ability to bind metals and affinity toward divalent cations like calcium cation has made them a suitable candidate for industrial as well as dentistry applications.

Phosphate containing compounds have found their way in underwater coatings, dental procedures, as fillers, drug delivery, bone implants and regenerative medicine [2].

Hydroxyapatite is used to carry out studies for phosphate-bone adhesion due to its resemblance to bone and its biocompatible nature. Thus, phosphorous functionalized

9 polymers can be used as an adhesive.

10 1.1. Adhesion

The term adhesion refers to the capability or tendency of two surfaces that are dissimilar in nature to clasp to each other whereas on the other hand cohesion refers to the clinging of two similar surfaces or particles to each other. Adhesion can be divided into several categories based on the intermolecular forces responsible for the adhesion and also the mechanism that it follows, categorized as below:

1.1.1 Mechanism of adhesion

Adhesion mainly depends on the interaction between the adhesive and the substrate in question and thus there are numerous factors that affect the adhesion of two:

- Surface tension and contact angle- This mechanism follows the process of interlocking the surfaces together by filling up the void or pores of the surfaces. It depends on the surface tension and the contact angle of the adhesive and the substrate. Surface energies of the substrate and the adhesive contribute to the wetting of the two against each other and the materials that wet against each other in general have a larger contact area as opposed to the ones that don’t. Wetting is defined as the potential of a liquid to interface with a substrate usually a solid. The extent of wetting is represented by contact angle (theta) measurement between the liquid and the substrate. Surface tension of the liquid along with the condition and nature of the substrate plays a vital role in determining the wetting ability. A smaller contact angle and low value of surface tension of the liquid results in an increased degree of wetting given that the surface is in a good condition i.e. cleaned. A wetted surface would have a contact angle less than 90 degree, the surface would have high surface energy and

11 the cohesive forces between the adhesive would be lesser than the adhesive force between

the adhesive and the substrate resulting in spreading of the liquid on the surface. On the other

hand for a contaminated surface (low surface energy) theta would be greater than 90 degree

and the cohesive forces in the adhesive would be greater than the adhesive forces between

the adhesive and the substrate, thus, resulting in dewetting of the liquid. Surface tension is

expressed in dyne/cm and is related to the contact angle by the following commonly used

equation:

γLA ·Cosθ =γSA –γSL

where, γLA is the liquid-air interfacial tension or liquid’s surface tension; γSA is the surface tension between solid-air approximating to the surface energy of the surface; γSL is the solid-liquid interfacial tension or surface tension between the solid and the liquid.

- Chemical adhesion- the mechanism of chemical adhesion involves bonding of the two

surfaces forming ionic, covalent or hydrogen bonds. The hydrogen bonds result in a

comparatively weaker bonding of the adhesive and the surface as compared to the

covalent and ionic bonds (fig.1.1). For two surfaces to adhere chemically they need to

be placed at a distance less than a nanometer from each other and hence these bonds are

fairly brittle.

Figure 1.1: Adhesive bond energy vs type of bonds

- Dispersive Adhesion- also known as physiosorption, it involves bonding of two

surfaces by vander Walls forces of attraction which is defined as attraction of

intermolecular forces. These forces can be weak or strong, the former known as

London force and the latter as dipole-dipole force of attraction. They are similar to the

bonds in chemical adhesion in terms of strength and thus a slight initiation of a crack

would result in its immediate propagation owing to the brittleness of the bond.

- Mechanical adhesion- also known as micromechanical adhesion, the adhesive in its fluid form flows through the crevices of the substrate and bonds to it through mechanical interlocking. Factors affecting this mechanism are the viscosity of the adhesive, attractive and repulsive intermolecular forces between the adhesive and the substrate and the thickness of the adhesive film.[7]

13 1.1.2. Cohesion- cohesion or cohesive force is the attraction of similar molecules towards each other.

In an adhesive it is the cohesive forces that binds the molecules of the adhesive together and adhesive forces on the other hand helps the adhesive bind on to a surface or substrate

(fig). Intermolecular and chemical bonds and interactions within the adhesive material are a few factors that affect the properties like the flow, viscosity and consistency of an uncured adhesive.

1.1.3. Failure modes in an adhesive joint

a) Structural failure-this kind of failure occurs when the substrate is weak and cannot withstand the forces applied by the adhesive or cohesive bonds, it thus shatters, cracks or breaks. https://foursevenfive.com/adhesive-bonds-and-failures-whats-going-on/ b) Adhesive failure- it occurs when the force exerted on the substrate is greater than the bond between the adhesive and the substrate, the adhesive is either partially or completely separated from the surface of the substrate (fig2). A surface with low surface energy or instability can cause such a failure. c) Cohesive failure- when the external force applied on an adhesive is greater than the cohesive bonds within it (fig1.2). Traces of adhesive can be seen on the substrate and this kind of failure is desired for.

Figure 1.2: Adhesive and cohesive forces acting in adhesion; adhesive and cohesivefailure

1.2. Lap shear test

This test is used establish the shear strength of an adhesive for a metal or a plastic substrate, ASTM D1002 and ASTM D3163 respectively. The test is conducted on single-lap joint specimen whose strength depends on the nature of the adhesive, thickness and nature of the metal and overlap area (fig.1.3a). The standard method uses an aluminum alloy sheet

25mm wide and 12.5mm overlap and varies depending on the test requirements. A universal tensile testing machine

15 (fig.1.3b) is used for the procedure which is conducted as follow:

- The shear area in sq inches or centimeters to which the sample is to be applied is measured.

The shear stress varies along the length of the joint for a simple lap joint.

- Each end of the specimen is then loaded on to the grip of the tensile testing apparatus.

- A force at a controlled is applied to the specimen until it breaks and maximum force is recorded

along with type of joint failure. Maximum shear stress is recorded in kilogram/square cm or

psi by dividing the maximum force by the shear area; type of failure can be adhesive or

cohesive.

For successful test results it is important that the surface of the sheet is decontaminated and smooth and the adhesive is cured on to it properly. Curing is performed at high temperatures (180 F) for 2-24 hours using any one of the following methods: Hot air ovens- these are used for large assemblies, slow in nature and depends on the thickness of the applied sample. Hot presses- uses steam or oil heated platens with temperature control mechanism and is used for large panels. Induction curing-it uses the resistance to the current flow in a conductive substrate caused by magnetic field and is used for fast curing.

Figure 1.3: a) Lap shear specimen; b) a tensile testing machine used for lap shear test

1.3. Adhesive technologies inspired by nature

Present day challenges to develop adhesives can be related to the adhesion problems of several

marine organisms. These organisms collect minerals and nutrients from their surroundings and

adhere to their surfaces with minimal or no separation on a day to day basis. These underwater

engineers have inspired scientists to mimic their mechanism for adhering in water.

1.3.1. Sandcastle worm: The sandcastle worm which is also known as honey tube worm is

a 3.0 inch long reef-forming polycheate and belongs to the family of sabellaridae. The name is

derived from their ability to build tube reefs analogous to sandcastles. The worm protects itself

physically by living in a shell composed of composite minerals and gathers the minerals

extrinsically in the form of sand grains and seashell hash bits rather than secreting them. These

minerals are bonded together by the worm into a tube with the help of small swabs of a

proteinaceous glue. Under cold water the glue is capable of setting under 30 seconds [3] and over a

period of several it toughens to give a leathery consistency. The glue consists of various acidic and basic proteins along with Mg2+ and Ca2+ ions [3]. The proteins are sequenced in a very simple and

repetitive manner, referred as Pc1-3. The composition of which can be described as follows- Pc1:

45 mol-% glycine, 14 mol-% lysine and 19 mol-% tyrosine, occurring in the form of 15 repeat

units of decapeptide VGGYGYGGKK. Pc2: dissipated copies of dodecapeptide

HPAVHKALGGYG. Pc3: consists of two major and several minor variants along with 4-13 serine

residues separated by single tyrosine residues. Amino acid studies of the glue have revealed that

serine and glycine constitute 50 mol-% of the total amino acid residues in the glue of which more

than 95% of the serine are phosphorylated[3,4] and approximately one-third of the tyrosine are in

the form of 3,4- dihydroxyphenyl-L-alanine (dopa) due to post-translational hydroxylation of the

18 tyrosine[6]. Dopa has proved to play a crucial role in interfacial adhesion. The physical attributes

like the foamy structure, fluidity and interfacial cohesion of the underwater adhesive from the worm can be explained by the complex coacervate theory. In aqueous solutions with low ionic strength the oppositely charged poly- electrolytes develop stoichiometric colloidal complexes that further results in the coacervate phase on reaching net neutrality. Hui shao et.al studied the application of coacervation effect to mimic adhesive based on glue from sandcastle worm. Mimic of the protein was synthesized using free radical copolymerization of acrylamide, dopamine methacrylate (DMA) and monoacryloxyethyl phosphate (fig.1.4) oxidizing dopa catechol to dopaquinone using NaIO4. Various ratios of primary amine were used to

synthesize lysine rich analogs. Both the analogs were mixed in different ratios and studied at different pH

value. An increase in coacervate density and bond strength observed with an increase in divalent cation ratios.

Figure 1.4: DOPA mimic using free radical copolymerization of acrylamide, dopamine methacrylate

1.3.2. Mussels: They belong to the marine family called Mytilidae and are most commonly found in

intertidal zones and attaches itself to the substrate with the help of tenacious byssals. They

reside more easily on rough surfaces with high energy as compared to smooth surfaces and

low energy and sense their target substrate by their foot tip which is extended from the gap

between the valves. The opening and closing of these valves is controlled by the adductor

muscles of the anterior and posterior portion. Once the mussel moves to the desired substrate

it attaches itself to the location by secreting proteins from its foot that forms a byssus

constituting of adhesive plaque, stem, root and thread. The roots act as the connector between

the retractor muscles inside the shells. The byssal plaque holds on to the substrate when the

foot goes back into the shell[5]. The byssal thread consists of an inner core that is flexible

and composed of polymerized collagen (precools) and matrix proteins(tmp) with a coating of a

thin but hardened layer of cuticle. The proteins in the byssal thread and plaque is composed

of mussel foot protein: mfp-1, mfp-2, mfp-3, mfp-4, mfp-5 and mfp-6. DOPA is the main

constituent of these proteins and contributes to the various functions of the each part of the

glue. B Kolbe et.al conducted a study to mimic mussel silk by combining catechol with

hydrophobic and electrostatic functional groups in a small molecule and were able to

synthesize a low molecular weight cateholic zwitterionic surfactant mimic (fig1.5).The study

showed the importance of maintaining a balance between the hydrophobic and electrostatic

functional groups and presence of catechol in order to optimize adhesion or coacervation.

Figure 1.5: synthesis of catechol with hydrophobic and electrostatic functional groups

1.3.3 Caddisfly

Caddisfly, also known as trichopetra is a freshwater aquatic species quite famous amongst fly fisherman habituating in cold water ranging from mountain streams to still marshes. It has over 12000 species broadly classified in three suborders namely The larvae of this species grows underwater where they feed and grow into a pupae which then flies temporarily to mate. The larvae feeds underwater with the help of a tubular structure that it uses to catch and store food and minerals. These larvae live in a well assembled stationery structure consisting of leaves, twigs etc held together by silk nets that enables it to capture food through a water channel.

Caddisfly produces its silk in the posterior and middle gland, the silk filament is comprised majorly of four blocks, SA,SB,SC and SD arranged orderly. The blocks further contains short but distinct motif with SA,SB and SC having almost a fixed number of amino acids, GPXG(G), GXXX, SXSXSX(SX)n whereas SD has a variable length due to a variable number of GPXGXXX repeats compared the silk produced by two different species of caddisfly with the silk produced by lepitopetra. The two species of caddislfly namely Hydropsche anngustpennis uses the silk to build the tubular structure whereas limniphelus decipiens use it to build protective structures to pupate respectively. The study showed the presence of H-fibroin and L-fibroin in the caddisfly silkand their interaction is similar to the fibroins present in lepitopetra. The l fibroin in both the species consisted of amino acid residues and cysteine and exhibited a 46% similarity with each other. The silk showed homology with the spacing in the lepidopetra silk sample. The h- 22 fibroin showed the presence of two non polar residues (Ile,Phe,Leu, Val, Pro) in groups bordered by charged residues(Asp, Glu,Lysp,Arg). The cys of all the three species was indispensable in order to produce proteins. However, they failed to find a homologue of p-

25(present in lepitopetra) in the caddisfly species. P-25 helps to form a beta sheet and provides mechanical strength and viscoelasticity in lepitopetra. The later secretes proteins from the posterior of the glands that is polymerized into a core filament which is then sealed into a to silk by the help of secricins produced at the middle section of the glands, the former on the other hand shows distinct secretions from the posterior and the middle glands.

The (SX) region is phosphorylated and thus forms the beta sheet structures insoluble in nature with the help of Ca2+

1.4. Role of phosphorous in adhesion

The presence of Phosphorous derived functional groups in phosphorous based polymers makes them an asset in various technological and medical applications. Baiping

Fu et.al. used FTIR, 31 P and ATR in order to understand the chemical mechanism through which phosphoric acid esters (PAEs) interact with hydroxyapatite which is a phosphate calcium ceramic biocompatible in nature resembling human bone . Self –etching primers incorporated with PAEs were used for the study and it was found these primers can chemically adhere and decalcify hydroxyapatite by the formation of water soluble PAEs- HA complexes. Findings by Subirade and Lebugle shows that PAEs substitute the hydroxyl group from the hydroxyapatite, thus forming mineral-organic linkages, owing to the

23 mechanism of adhesion of the two. Such self-etching primers are often used in dentistry applications. Veronique et.al. studied the affinity of phosphorous containing cyclodextrin polymers (CDP) towards hydroxyapatite and metal cations. The study showed good binding affinity of CDP to divalent magnesium, zinc and calcium cations which increased on increasing the molecular weight of CDPs exhibiting the ability to form complexes towards amphiphilic and divalent cationic species.

24 Chapter II

SYNTHESIS AND CHARACTERIZATION OF N-SUBSTITUTED DIOLS

2.1. Introduction

This chapter discusses about the synthesis of N-substituted diols including serine

mimic monomer, catechol mimic monomer and soy mimic monomer and their chemical

composition characterization by 1H NMR.

2.2. Instrumentation

The instruments used to characterize the monomers are as follows, also mentioned is

the procedure followed to do the same:

1H NMR spectroscopy was used to characterize the chemical composition of the

synthesized monomer in this chapter using a VARIAN 300 mHz NMR instrument. ACD

NMR processor was used to analyze the obtained data.

2.3 Materials

Diethanolamine (99%) was purchased from Alfa Aesar. Methanol, Hexane was

bought from the chemical store of the University of Akron. Dichloromethane was purchased

from fisher scientific and dried in our lab. Trimethylamine, 4-hydroxy butyric acid methyl

ester, Tert-butyldimethylchlorosilane was purchased from VWR.

25 2.4. Synthesis of serine mimic monomer

To a single neck round bottom flask with a stir bar of 4-hydroxy butyric acid methyl

ester (59.32 mmol, 7 grams), anhydrous dichloromethane (DCM,60mL), Et3N (130.50

mmol, 8.884 grams) was added under nitrogen and ice cooling condition. Tert-

butyldimethylchlorosilane (65.23 mmol, 9.835 mL) was then added to the flask using a

syringe. The reaction was allowed to stir for half an hour in an ice bath and stirred overnight

at room temperature. The product was dissolved in 100mL DCM and transferred to a

separating funnel along with water (50mL). The organic layer was collected and allowed to

dry using anhydrous Na2 SO4. The product was concentrated by rotational evaporation and

purified by running a column using 10% EtOAc in Hexane, concentrated by rotational

evaporation and keeping it on high vacuum for overnight. 90% yield was obtained.

Scheme 1: Synthesis scheme for serine mimic monomer

1H NMR (300MHz, Chloroform –d)

The obtained and diethanolamine was then added to a single neck round bottom

flask with a stir bar and allowed to reflux overnight at 80 degree Celsius. The product was

purified by running a silica gel column with 5% methanol in DCM. Solvent was removed

by rotational evaporation and dried by keeping it at high vacuum line overnight.

26 2.5 Synthesis of catechol mimic monomer

To a single neck round bottom flask with magnetic stir bar diethanolamine

(8.46mmol, 2grams) and (12.69mmol, 1.33grams) was added and heated at 100 degree

Celsius for 24 hours under vacuum. A red coloration was observed after the overnight reaction. The reaction mixture was diluted in EtOAc and washed with acid and brine solution.

Scheme 2: synthesis of catechol mimic monomer

The organic layer (yellow in color) was collected and dried to obtain yellow colored particles. The product was purified by flash column chromatography with trichlorometahne to remove dietnaolamine from the product. A yield of 2.3grams was obtained.

2.6 Synthesis of soy mimic monomer

To a single neck round bottom flask along with a magnetic stir bar diethanolamine

(143.538mmol, 15.091grams) and sodium methoxide (11.961mmol, 6.461grams) were added and allowed to homogenize. Once homogenized, soy bean oil (23.92mmol, 2grams) was added dropwise using an additional funnel. The reaction mixture was allowed to stir overnight at 110 degree Celsius under vacuum. Methanol was then added after 15minutes 27 of stopping the reaction. The obtained product was washed with EtOAc and brine solution, collecting the organic layer. Product was purified by column chromatography with 5%

MeOH in DCM.

Scheme 3: synthesis of soy mimic monomer

28 CHAPTER III

NMR RESULTS AND DISCUSSION OF THE N-SUBSTITUTED DIOL MONOMER MIMIC

3.1. Introduction

This chapter will discuss the 1H NMR data obtained and the analyses of the peaks

for each NMR resulting in the synthesized monomer.

3.2. Instrumentation

1H NMR spectroscopy was used to characterize the chemical composition of the

synthesized monomer in this chapter using a VARIAN 300 mHz NMR instrument. ACD

NMR processor was used to analyze the obtained data.

3.3. 1H NMR peak data

Resonance of the catechol functionalized group was observed at 1.65 ppm (fig 3.1)

and the resonance for the aliphatic chain, double bond of the soy mimic monomer was

observed at 5-5.5 ppm (fig. 3.2) and the resonance for TBDMS group was observed at 0.09

ppm (fig.3.3). Thus, the monomers were synthesized successfully.

Figure 3.1: 1H NMR of catechol mimic monomer

Figure 3.2: 1H NMR of soy mimic monomer

Figure 3.3: 1H NMR of serine mimic monomer

31 CHAPTER IV

SYNTHESIS OF POLYESTERS DERIVED FROM N-SUBSTITUTED DIOLS

MONOMER MIMICS

4.1 Introduction

This chapter will discuss the synthesis and characterization of polymers derived from the

mimic monomers prepared in the previous chapter. NMR results of the polymer and its

adhesive strength will be discussed along with the procedure for the same.

4.2 Materials

Sebacic acid, diethylchlorophosphate, t-baf, TIPS, bromotrimethylsilane were purchased

from ALFA AESAR. Hexane and methanol was bought from chemical store of the

university , DCM was purchased from fisher scientific and dried in the lab, DIC, DPTS was

purchased from ALDRICH.

4.3 Synthesis of the Parent Polymer

To a two neck round bottom flask with a magnetic stir bar the obtained soy monomer

(5.492mmol, 2.019grams), serine monomer (1.569mmol, 4.79grams) and catechol monomer

(0.784mmol, 2.427grams) and DPTS (3.138mmol, 9.239 grams) were added keeping the

temperature at 0 degree Celsius and purging nitrogen for around ten minutes. Anhydrous

DCM (12 mL) was then added to the reaction mixture and homogenized, DIC (31.8 mmol,

4.95 mL) was added dropwise at 0 degree Celsius using a syringe under nitrogen. The

reaction mixture was then allowed to stir for 48 hours at room temperature. The obtained

product was diluted in DCM and kept in the refrigerator for approximately 15 minutes

followed by filtration using a Buchner funnel. The product was precipitated in DCM and

32 MeOH (x2). The resultant product was dissolved in DCM and dried using rotational

evaporation and kept under high vacuum overnight to dry. Yield 55%

t-BAF

(i) Diethylene cholorophosphate, imidazole

(ii) SiMeBr3 (iii) TFA, TIPS

Scheme 4: synthesis route from the parent polymer to the polyester adhesive

4.4 Deprotection of TBDMS

The obtained polymer was dissolved in DCM (12 mL) and transferred to a two neck

round bottom flask with magnetic stir bar under nitrogen. 5 mL of t-Baf was added to the

flask kept in an ice bath under nitrogen. The reaction was allowed to stir for 2 hours under

nitrogen.

33 4.5. Phosphorylation of the de protected TBDMS

The round bottom flask containing the deprotected polymer product was kept under

vacuum cycle for ten minutes and was then dissolved in anhydrous DCM (12 mL). N-

methylimidazole (1 mL) was then added dropwise using an additional funnel under an ice

bath and nitrogen. Diethylene chlorophosphate (1mL) was then added dropwise under ice

bath and nitrogen. The ice bath was removed after 15 minutes and the reaction mixture

was allowed to stir for an hour. Separation was done by adding a saturated solution of

ammonium chloride and DCM (x2), the organic layer was collected and dried by adding

anhydrous sodium sulfate for 15 minutes. Solvent from the obtained product was removed

by rotational evaporation and was kept for drying under high vacuum overnight.

4.6. De-protection of the ethyl ether bond

The product from phosphorylation was dissolved in anhydrous DCM (10 mL) and

transferred to a two neck round bottom flask with stir bar and purged with nitrogen and

kept under vacuum – nitrogen cycle for ten minutes. SiMe3Br (5 mL) was then added to

the flask dropwise using an additional funnel under ice bath and nitrogen. The reaction

was allowed to stir for two hours at room temperature. Solvent from the product was

removed using rotational evaporation and kept under high vacuum for half an hour.

Methanol (12 mL) was then added to the product and allowed to stir for an hour. The

product was dried by rotational evaporation and kept for drying under high vacuum

overnight. A dark yellow and viscous product was observed.

4.7. De-protection of the catechol group

The polymer obtained from the above step was dissolved in anhydrous THF (12 mL) and transferred to a two neck round bottom flask with a stir and kept under nitrogen – vacuum cycle for ten minutes. TIPS (1 mL) was then added to the flask dropwise under ice bath and nitrogen. TFA (1 ML) was Then added dropwise to the reaction mixture under ice bath and nitrogen. The reaction was allowed at room temperature for 1 hour. The obtained product was precipitated using DCM and brine solution (x2). The organic layer was collected and the contents were dried using rotational evaporation technique and kept at high vacuum for overnight. Yield(95%)

35 CHAPTER V

CHARACTERIZATION AND ADHESIVE STRENGTH OF THE POLYESTER

5.1 Introduction

This chapter will discuss the 1H NMR analyses of the data obtained and the

procedure for lap shear test along with the result obtained from the same.

5.2 Lap shear measurement of the polymer

Lap shear testing of the obtained polymer was done on aluminum substrate using

universal tensile testing apparatus. Procedure for testing is as follows:

Firstly, aluminum substrate 8.0 cm long, 1.20 cm by breadth was made to order. Before

starting the test the substrate was cleaned using hexane, acetone and methanol, by

immersing the substrate in a beaker filled with each solvent and sonicating it for half an

hour each and then drying it in an oven for an hour. The obtained polymer was transferred

into different vials with varying percentages of tetrabutylammonium periodate (4-20% of

the polymer). For each sample 5 lap shear joints were prepared: 12 mg of each sample was

spread at an area of 1.5 cm from the edge of the aluminum substrate, a second substrate

plate was then carefully placed over the one containing the sample making sure that it

spreads evenly and clamped together. The joint was then left for curing at two different

temperatures: 25 and 50 degree Celsius also varying the days of curing (1, 3 and 5 days).

5.3. Results and Discussion

1H NMR data analyses of the protected parent polymer

Resonance of the double bonds present in the aliphatic chain of the soybean mimic

monomer was observed at 5.33 ppm, resonance of the aromatic group of the catechol was 36 observed at 6.61 ppm, and resonance for the protected catechol could be seen at 1.84

ppm and for the protected TBDMS group resonance was observed at 0.04. The NMR

result showed the successful synthesis of the protected parent polymer.

1H NMR data analyses of the TBDMS de-protected polymer:

Resonance peak for the OH group of the de-protected TBDMS was observed at 4.20 ppm

and the absence of the peak at 0.04 ppm was observed. Thus, the TBDMS group was

successfully de-protected.

1H NMR data analyses of the phosphorylated polymer:

Resonance peak for the protected ethyl ether group was observed at 1.31 and 4.10 ppm.

1H NMR of the de-protected ethyl ether group:

De-protection of the ethyl ether group was confirmed by the absence of the peaks at 1.31

and 4.10 ppm, thus observing a successful de-protection of the same.

1H NMR data analyses of the de-protected catechol :

De-protection of the catechol group was confirmed by the absence of the peak at 1.69 ppm.

As per the NMR analyses a polyester containing phosphorylated serine was

successfully synthesized in line with the proposed scheme of synthesis.

Figure 5.1: 1H NNMR spectra for the parent polymer

Figure 5.2: 1H NNMR spectra for the TBDMS deprotected polymer

Figure 5.3: 1H NNMR spectra for the phosphorylated polymer

Figure 5.4: 1H NNMR spectra for the deprotected ethyl ether group

Figure 5.5: 1H NNMR spectra for the deprotected catechol group

b c

Figure 5.6: Lap Shear results of the synthesized polymer; a) Lap shear strength vs Polymer to periodate ratio; b) Lap shear strength vs temperature; c) lap shear strength vs days

43 The lap shear strength of the polymer was observed to decrease on increasing the polymer-iodate ratio, however an optimum value was observed (fig.5.6a), an increase in the strength was observed on curing the joint at 25 degree Celsius (fig 5.6b), however there was no significant change in strength observed on varying the days of curing from 1 vs 5 days (fig 5.6 c)

44 CHAPTER VI

CONCLUSION

In this work a synthetic strategy to mimic the adhesion mechanism of an underwater inspired form the previous work done in this field by functionalizing the N-diols and incorporating them into a system. The adhesive strength of the synthesized mimic polymer was measured by lap shear testing.

A successful caddisfly mimic polymer adhesive was achieved with the help of a library of N- substituted diols available in the lab. A solvent free system for easy application of the adhesive was achieved by using higher ratio of soy mimic monomer. It was observed that the catechol mimic monomer played a crucial role in providing cohesive strength whereas the phosphorylated monomer was responsible for interfacial strength of the synthesized adhesive. A UV free crosslinking system was achieved for adhesive strength of the polymer and the cohesive strength was obtained from the catechol group inspired from DOPA present in mussels, another underwater engineer.

The NMR data showed successful deprotection of the pendant groups and phosphorylation, which plays a crucial role in adhesion was observed. An optimum value for polymer-crosslinker ratio and the conditions required for curing of the polymer was established.

REFERENCES

1) International Journal of Dentistry Volume 2012 (2012), Article ID 951324

2) Ashton, N. N.; Taggart, D. S.; Stewart, R. J. Biopolymers 2012, 97

3) Sehnal, F.; Akai, H. Int. J. Insect Morphol. Embryol. 1990, 19, 79

4) Yonemura N, Sehnal F, Mita K, Tamura T. Biomacromolecules. 2006 Dec;7(12):3370-8.

5) Wang, Y.; Sanai, K.; Wen, H.; Zhao, T.; Nakagaki, M. “Characterization of unique heavy

chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata

(Trichoptera; Stenopsychidae)”. Mol. Biol. Rep. 2010, 37 (6), 2885−92

6) Naoyuki Yonemura, Frantis ˇek Sehnal, Kazuei Mita, and Toshiki Tamura. “Protein

Composition of Silk Filaments Spun under Water by Caddisfly Larvae”.

Biomacromolecules 2006,7, 3370- 3378

7) JanuszW.Strzelecki1,JoannaStrzelecka2,KarolinaMikulska1,MariuszTszydel3,Aleksander

Balter1,WiesławNowak1.“Nanomechanics of newmaterials–AFM and computer modelling

studies of trichoptera silk”. Cent. Eur. J.Phys.,9(2),2011.482-491.

8) J. Herbert Waite , Niels Holten Andersen , Scott Jewhurst & Chengjun Sun (2005) “Mussel

Adhesion: Finding the Tricks Worth Mimicking”, The Journal of Adhesion, 81:3-4, 297-317,

DOI: 10.1080/00218460590944602

9) František Sehnal1 and Tara Sutherland. “Silks produced by insect labial glands”. [Prion 2:4,

46 10) Hui Shao, Kent S Bachus and Russel J. Stewart. “ A water borne adhesive modelled after

Sandcastle Glue” Macromol. Biosci.2009,9,464–47,2009 WILEY-VCH Verlag GmbH &

Co. KGaA, Weinheim

11) Wang, C.-S.; Pan, H.; Weerasekare, G. M.; Stewart, R. J. Peroxidase-catalysed interfacial

adhesion of aquatic caddisworm silk. J. R. Soc., Interface 2015, 12 (112), 20150710

12) Zhou,J.;Defante,A.P.;Lin,F.;Xu,Y.;Yu,J.;Gao,Y.;Childers,E.;Dhinojwala,A.;Becker,M.L.Ad

hesi on propertiesofcatechol-based biodegradable amino acid-based poly(ester urea)

copolymers inspired from mussel proteins. Biomacromolecules 2015, 16 (1), 266−74.

13) MacGillivray, T.E. Fibrin sealants andglues. J.CardSurg 2003,18 (6), 480−5.

14) Fu,B.;Sun,X.;Qian,W.;Shen,Y.;Chen,R.;Hannig,M.Evidence of chemical bonding to

hydroxyapatite by phosphoric acid esters. Biomaterials 2005, 26 (25), 5104−5110.

15) Zeller, A.; Musyanovych, A.; Kappl, M.; Ethirajan, A.; Dass, M.; Markova, D.; Klapper,

M.; Landfester, K. Nanostructured coatings by adhesion of phosphonated polystyrene

particles onto titanium surface for implant material applications. ACS Appl. Mater.

Interfaces 2010, 2 (8), 2421−8.

16) Watson, B. M.; Kasper, F. K.; Mikos, A. G. Phosphorouscontaining polymers for

regenerative medicine. Biomed. Mater. 2014, 9 (2), 025014.

17) Datta, P.; Chatterjee, J.; Dhara, S. Electrospun nanofibers of a phosphorylated polymerA

bioinspired approach for bone graft applications. Colloids Surf., B 2012, 94, 177−183.

18) Stewart, R. J. Protein-based underwater adhesives, the prospects for their biotechnological

production. Appl. Microbiol. Biotechnol. 2011, 89 (1), 27−33.

47 19) Yonemura, N.; Mita, K.; Tamura, T.; Sehnal, F. Conservation of silk genes in Trichoptera

and Lepidoptera. J. Mol. Evol. 2009, 68 (6), 641−53.

20) Freeman supply datasheets, “an adhesive guide”

21) adhesives-sealants; science-of-adhesion;adhesion-cohesion.

22) Wilker, J. J. Angew. Chem. Int. Ed. 2010, 49, 8076-8078 (ii) Ahn, B. K. et al. Nat. Commun.

2015, 6, 8663 (1-7)

23) Kollbe Ahn, B.; et al. J. Am. Chem. Soc. 2015, 137, 9214−9217