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Investigation into the control of melittin secondarystructure and antimicrobial activityZachary B. MolinetsPurdue University

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Recommended CitationMolinets, Zachary B., "Investigation into the control of melittin secondary structure and antimicrobial activity" (2014). Open AccessTheses. 656.https://docs.lib.purdue.edu/open_access_theses/656

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Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared

By

Entitled

For the degree of

Is approved by the final examining committee:

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Zachary B. Molinets

INVESTIGATION INTO THE CONTROL OF MELITTIN SECONDARYSTRUCTURE AND ANTIMICROBIAL ACTIVITY

Master of Science

Jean A. Chmielewski

Arun K. Ghosh

Christine A. Hrycyna

Jean A. Chmielewski

R. E. Wild 7/17/2014

INVESTIGATION INTO THE CONTROL OF MELITTIN SECONDARY

STRUCTURE AND ANTIMICROBIAL ACTIVITY

A Thesis

Submitted to the Faculty

of

Purdue University

by

Zachary B. Molinets

In Partial Fulfillment of the

Requirements of the Degree

of

Master of Science

August 2014

Purdue University

West Lafayette, Indiana

ii

For my wife, Natalie, who has helped me grow and open my mind,

since the first time we said hello.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES ...............................................................................................................v

LIST OF FIGURES ........................................................................................................... vi

LIST OF SCHEMES........................................................................................................ viii

ABSTRACT ....................................................................................................................... ix

CHAPTER ONE ANTIMICROBIAL PEPTIDES ..............................................................1

Introduction ....................................................................................................................1 Antimicrobial Peptides...................................................................................................2

Sources of Antimicrobial Peptides...........................................................................4 Antimicrobial Peptides with α-helical Secondary Structure ..........................................6

Mechanisms of Action .............................................................................................6 Metal Stabilized/Destabilized Peptides..........................................................................9 Siderophores ................................................................................................................11 References ....................................................................................................................13

CHAPTER TWO MELITTIN ANALOGUES ..................................................................16

Melittin .........................................................................................................................16 Activity of Melittin ................................................................................................18 Structural Requirements for Activity .....................................................................19

Truncated Melittin .......................................................................................................20 1st Generation Melittin Derivatives ..............................................................................21

Synthesis of Metal Binding Moieties ...............................................................23 Synthesis of Melittin Derivatives.....................................................................24 Circular Dichroism...........................................................................................25 Antimicrobial Activity .....................................................................................27

2nd Generation Melittin Derivatives .............................................................................27 Synthesis of 2nd Generation Melittin Derivatives ...........................................28 UV/Vis Spectroscopy.......................................................................................29 Circular Dichroism...........................................................................................30 Antimicrobial Activity .....................................................................................33

iv

Page

Reversibility of the Metal-Ligand Interaction .............................................................34 Melittin Derivatives with Other Metals .......................................................................35 Conclusions ..................................................................................................................37 Materials and Methods .................................................................................................37 Experimental ................................................................................................................38 References ....................................................................................................................43

v

LIST OF TABLES

Table Page

1.1 Amino acid sequences of cecropin-A, magainins-II, protegrin-1, melittin and defensins. Subscript numbering indicates disulfide bridge ............................................4

1.2 Examples of antimicrobial peptides isolated from invertebrates. ..................................5

2.1 Purification of melittin derivatives ..............................................................................25

2.2 Purification of truncated melittin derivatives ..............................................................29

2.3 MIC of melittin derivatives ..........................................................................................34

vi

LIST OF FIGURES

Figure Page

1.1 The barrel-stave representation of AMP activity ...........................................................7

1.2 Toroidal Pore representation of AMP activity ...............................................................8

1.3 The “carpet model” representation of AMP activity .....................................................9

1.4 Generic profile of an α-helix highlighting positions of interaction. ............................10

1.5 Structures of enterobactin(top), pyochelin(bottom left), and rhodotorulic acid(bottom right) ........................................................................................................12

2.1 The structure of melittin...............................................................................................16

2.2 Helical wheel diagram of melittin ................................................................................17

2.3 3D representation of melittin showing the “bent rod” .................................................18

2.4 The structure of truncated melittin(12-25). ..................................................................21

2.5 Placement of metal binding moieties to melittin, where X represents bipyridine or terpyridine ..............................................................................................21

2.6 Metal assisted deactivation of peptide with siderophore reactivation .........................22

2.7 CD spectra of melittin derivatives, 50 μM, in 100 mM phospate buffer, pH 7. ..........26

2.8 CD spectra of melittin derivatives, 50 μM, in 100 mM phospate buffer, pH 7.4, with 1 equivalent Fe(II) ........................................................................................26

2.9 E. coli antibacterial assay of melittin derivatives. 6 hour incubation ..........................27

2.10 Second generation melittin derivatives. From top to bottom: MLTx, MLTx1, MLTx2, MLTx3. Where X is bipyridine or terpyridine ..............................................28

2.11 UV/Vis spectroscopy of MLTx1-tpy:Fe(II), 100 μM, in water .................................30

vii

Figure Page

2.12 UV/Vis spectroscopy of MLTx3-tpy:Fe(II), 100 μM, in water .................................30

2.13 CD of melittin derivatives, 50 μM, in 10 mM phosphate buffer, pH 7.4 ..................32

2.14 CD of melittin derivatives, 50 μM, in 10% HFIP/water ............................................32

2.15 CD of melittin derivatives, 50 μM, with metal ion in 10 mM phosphate buffer, pH 7.4. MLTx3d-tpy was denatured prior to metal addition ...........................33

2.16 MLTx3-tpy with 1 equiv Fe(II) in solution. 500 µM solution of the peptide-Fe(II) complex with 2 equivalents of chelators ...........................................................35

2.17 UV/Vis spectrum of MLTx3-tpy:Co(II), 100μM, (blue) and terpyridine control (red) .................................................................................................................36

2.18 CD of melittin derivatives, 50 μM, in 10 mM phosphate buffer, pH 7.4 ..................36

viii

LIST OF SCHEMES

Scheme Page

2.1 Synthesis of metal binding ligands ..............................................................................23

2.2 Synthesis of melittin derivatives, where X is terpyridine or bipyridine ......................24

ix

ABSTRACT

Molinets, Zachary B. M. S., Purdue University, August 2014. Investigation Into the Control of Melittin Secondary Structure and Antimicrobial Activity. Major Professor: Jean A. Chmielewski.

Antimicrobial resistance has been an exponentially growing problem since the

discovery of antibiotics. Antibiotics have been misused for many years and this misuse

has grown into a real problem for the medical community. While there are countless

safeguards to prevent infection by a resistant strain of bacteria, there are still many

plagued by it and must be treated with sometimes dangerous antibiotics. Melittin, along

with many other peptides, contain potent antimicrobial properties, but are also toxic

toward enthrocytes. The control of the secondary structure of peptides provides the key to

adjusting their activity.

1

CHAPTER ONE ANTIMICROBIAL POLYPEPTIDES

Introduction

Antimicrobial resistance has been an exponentially growing problem since the

discovery of antibiotics. The misuse of antibiotics is a major driving force for antibiotic

resistance. The first source of misuse was during the golden age of antibiotics, when there

was very little resistance. Antibiotics would be prescribed for the slightest symptoms,

with this wide spread use bacteria found ways to adapt and resist the drugs.1 Another

source of misuse is in medical patients, when a prescription is not taken as directed.

Patients across the globe, especially in less developed countries, have said that they save

or do not finish the prescription.2 Overuse in agriculture is another major source of

resistance. In 2010 the FDA released a report estimating that the use of antibiotics in food

animals was over 11 million kilograms were used in 2009, and 2011 the number has rose

to 13.5 million kilograms.3 After bacteria encounter a class of antibiotics over several

generations, the antibiotic becomes less effective and resistance is born.

Resistance is formed by mutations in the bacterial DNA that evolve mechanisms

to counter antibiotics. β-lactams are a class of antibiotics which block a pathway in the

production of the cell wall, thereby killing the bacteria. β-lactams such as penicillin or

amoxicillin are the workhorse of antibiotics and are the most prescribed antibiotics

available. As such, the resistance to β-lactams has risen dramatically. The mechanism of

2

bacterial resistance has been pinpointed to β-lactamase enzymes. These enzymes cleave

the lactam ring making the β-lactam compounds inert. There are several groups of β-

lactamase enzymes, each are capable of a range of mechanisms. For example, some β-

lactamase enzymes are capable of being inhibited by clavulanic acid, a β-lactam mimetic.

However, there are also some that show resistance to clavulanic acid. These data point to

the necessity for novel strategies to combat resistance.

There is not only a biological cost to the increase in bacterial resistance but a

societal cost as well. Increasing resistance brings with it increased mortality and health

care expenses. For salmonella, a fairly common source for food poisoning, the mortality

rate against resistant strains is 3.4%, while only 0.2% for non-resistant strains.4 The

increased patient cost to treat resist strains of bacteria in the United States is greater than

8 billion US dollars.5

Antimicrobial Peptides

Antimicrobial resistance from misuse of antibiotics has created a global health

concern which draws the need for novel types of antimicrobial therapeutics.

Antimicrobial peptides are short, 15-40 amino acids, peptides that are a part if the innate

immune response in organisms.6 Many isolated AMPs have been shown to have activity

against a wide array of organisms including Gram-positive and Gram-negative bacteria,

fungi, and viruses.7 The diverse mechanisms of activity or AMPs include formation of

pores in the membrane, inhibiting proteins, and triggering autolysis.6 Antimicrobial

3

peptides are attractive as a new class of clinical antibiotics because of their broad

spectrum of activity and diverse mechanisms in contrast to small molecule antibiotics.8

Antimicrobial peptides (AMPs) first came into light in the 1980’s when Boman,

Zasloff, and Lehrer concurrently isolated AMPs from separate animal kingdoms.9,10

Notably, in Zasloff’s lab, it was noticed that infections rarely occurred after non-sterile

surgical procedures with the African clawed frog Xenopus laevis.11 It was discovered that

small peptides in the skin had broad spectrum antibacterial properties, and these peptides

became known as magainins.11 Since the discovery of the first AMPs over 800, have been

found throughout the animal and plant kingdom.12

There are several inherent characteristics associated with AMPs. AMPs are

usually short, less than 50 amino acids, and contain a cationic amphipathic structure.13

AMPs are classified by their amino acids and secondary structure: α-helix, β-sheet,

cysteine containing, containing many standard AA, and containing modified AAs.7

Cecropins, one of the first classes identified, is a cationic amphipathic peptide isolated

from Hyalophora cecropia.14 Through NMR studies it was determined that cecropins

form an α-helix in 15% HFIP.15 Peptides which form α-helical secondary structures are

most known for their pore forming mechanism. Some well studied peptides with this

mechanism are cecropins16, melittin17 magainins18, and protegrin-1.19

4

Table 1.1: Amino acid sequences of cecropin-A, magainins-II, protegrin-1, melittin and defensins. Subscript numbering indicates disulfide bridge.

Peptide Sequence

Cecropin-A KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2

Magainin-II GIGKFLHSAKKFGKAFVGEIMNS

Protegrin-1 RGGRLC1YC2RRRFC2VC1VGR-NH2

Melittin GIGAVLKVLTTGLPALISWIKRKRQQ-NH2

Human α-

defensin ACYCRIPACIAGERRYGTCIYQGRLWAFCC

Sources of Antimicrobial Peptides

As stated earlier, the first AMPs isolated were from amphibians and many more

AMPs have since been isolated from a plethora of species, including insects, plants,

animals, and humans. Additionally, these peptides have a broad range of secondary

structures and activity against different types of invasive organisms.20 This menagerie of

structure, activity, and chemical properties is a hotspot for the development of novel

therapeutics.

AMPs in invertebrates were first isolated from Hyalophora cecropia, commonly

known as the Cecropia moth, the largest moth in North America.21 These peptides

became known as ceropins and have broad spectrum activity against protozoa, bacteria,

and fungi.7 Since the initial discovery of AMPs of the Cecropia moth several unique

AMPs have been identified in many other invertebrates ranging from honey bees to crabs.

5

A sample of some of those peptides can be seen in table 1.2 with their respective

activities.

Table 1.2: Examples of antimicrobial peptides isolated from invertebrates

Source Peptide Activity

Drospohila melanogaster Drosomycin F

Diptericin G-

Aedes Aegypti Defensin-α G-,G+

Defensin-β G-,G+

Apis mellifera Mellitin G-,G+, H

Defensin G-,G+

Royalisin G-,G+

Lycosa carolinensis Lycotoxin G-,G+

Stomoxys calcitrans Smd1 G-,G+

Smd2 G-,G+

Tachypleus tridentatus Tachyplesin G-,G+

Limulus polyphemus Tachyplesin II G-,G+,F

Polyphemnsin I G-,G+,F

Polyphemnsin II G-,G+,F

Androctonus australis Androctonin G-,G+,F

Mytilus edulis Mytilin A G-,G+

Hyalophora cecropia Cecropin G-

G-, Gram Negitive; G+, Gram Positive; F, antifungal; H, Hemolytic

With the initial discovery of AMPs in amphibians and soon invertebrates, it

wasn’t long until AMPs were being discovered in many other species including plants

and other vertebrates. Defensins are a group of AMPs that are found in several different

mammals including humans and rabbits, and they also have been isolated from plants and

insects.22 Defensins are 41 amino acids in length and are characterized by their disulfide

6

bridges. Defensins are classified into sub-categories depending on the arrangement of the

disulfide bridges.

Antimicrobial Peptides with α-helix Secondary Structure

Particular classes of AMPs are highly predisposed to form pores in plasma

membranes. α-helical AMPs are one such class that lyses pathogens and erythrocytes. An

α-helical AMP's first interaction with a target is electrostatic due to the cationic amino

acids (AAs) interacting with the negatively charged heads of the phospholipids in the

plasma membrane or other negatively charged components on the cell wall. There is

much speculation about the mechanism of pore forming peptides, but the consensus is

that three types of mechanisms exist: toroidal pore, carpet model, and barrel stave.23

Mechanism of Action

The barrel stave model, figure 1.1, describes a mechanism where the peptides

integrate into the plasma membrane and form a pore. The amphiphatic characteristics of

the peptide make this possible in that the hydrophobic features of the peptide is faced

outward toward the hydrophobic tails of the membrane and the hydrophilic face forms an

aqueous pore.24 One peptide alone cannot form the aqueous pore, therefore, many

peptides come together to form the pores. Some AMPs, such as melittin, utilize a

structural feature known as the leucine zipper to hold the pore together.25 The stability of

the hydrophobic interactions and the leucine zipper allow for the intercellular material to

leak through the pore from the cell.

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11

the Shultz laboratory synthesized model peptides and incorporated a disulfide bridge at

the i, i + 7 positions.32 Taylor incorporated amide bonds into model peptides utilizing the

natural functional groups of lysine and aspartic acid or glutamic acid.33 The formation of

the amides stabilized the helical structure when compared to the acyclic analogue. While

these methods could provide triggers, as with the disulfide bonds, or permanent

modifications with amide bonds, more reversible methods could provide great value.

Metal – ligand interactions allow a lot of variability and exploration with various

metals. For example, Paul Hopkins’ laboratory used various divalent metal ions to

stabilize the helicity of model peptides.34 The study found that Co(II), Ni(II), Cu(II),

Zn(II), and Cd(II) all had a positive effect with most of the peptides. In another study by

Futaki et al, the group was able to stabilize or destabilize model peptides using

iminodiacetic acid.34 The group made another step to selectively destabilize the Jun-Fos

herterodimer to form a Jun-Jun homodimer.31 The destabilizing of one peptide in the

heterodimer changes the natural recognition of the dimer.

Siderophores

Iron is a critical metal in many organisms as it is needed to do a range of activities

in different enzymes. In mammals iron is needed to transport oxygen in the blood and is a

cofactor of superoxide dismutase which neutralizes harmful radicals in the body. Iron is

also required by bacteria. For example, in E. Coli it is a cofactor in 3-deoxy-D-

arabinoheptulosonate 7-phosphate (DAHP) synthase which is used to make aromatic

compounds and is in the biosynthetic pathway to produce the aromatic amino acids.35

12

Bacteria obtain iron by releasing a class of compounds known as siderophores. These

compounds have some of the highest known binding constants to iron(III). For example,

enterobactin, is the siderophore released by E.Coli for iron scavenging. Enterobactin

binds to iron with the catechol moieties and is brought into the cell by transport proteins

which have receptors on the cell surface.36 Rhodotorulic acid is another siderophore

isolated form Rhodotorula pilimanae.37 Unlike enterobactin which is a catechol based

siderophore, rhodotorulic acid is a hydroxymate. Additionally, rhodotorulic acid contains

only four binding sites, a complex of Fe2(RA)3 must be formed. Pyochelin is another

siderophore isolated from Pseudomonas aeruginosa.38 Pyochelin is unique in its structure

compared with other siderophore which are usually rich in catechols or hydroxymates.

Figure 1.5: Structures of enterobactin(top), pyochelin(bottom left), and rhodotorulic acid(bottom right).

13

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14

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27) Matsuzaki, K., Murase, O., Fujii, N, Miyajima, K., An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation. Biochemistry, 1996, 35, 11361-11368

15

28) Yamaguchi, S., Huster, D., Waring, A., Lehrer, R., Kearney, W., Tack, B., Hong, M., orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state nmr spectroscopy. Biophysical Journal, 2001, 81, 2203–2214

29) Shai, Y., Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by K-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochimica et Biophysica Acta, 1999, 1462, 55-70

30) Henchey, L., Jochim, A., Arora, P., Contemporary strategies for the stabilization of peptides in the a-helical conformation Current Opinion in Chemical Biology 2008, 12, 692–697

31) Futaki, S., Kiwada, T., Sugiura, Y., control of peptide structure and recognition by Fe(III)-induced helix destabilization. J. Am. Chem. Soc. 2004, 126, 15762-15769

32) Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G. General approach to the synthesis of short α-helical peptides. J. Am. Chem. Soc. 1991, 113, 9391-9392

33) Osapay, G.; Taylor, J. W. Synthesis and conformational properties of a highly α-helical uncosapeptide constrained by three side-chain to side chain lactam bridges. J. Am. Chem. Soc. 1992, 114, 6966-6973

34) Ruan, F.; Chen, Y.; Hopkins, P. B. Metal ion enhanced helicity in synthetic peptides containing unnatural , metal ligand residues. J. Am. Chem. Soc. 1990, 112, 9403- 9404

35) Messenger, A., Barclay, R., Bacteria, iron, and pathogenicity. Biochemical Education., 1983, 11, 54-63

36) Neilands, J., Siderophores: structure and function of microbial iron transport compounds. J. Bio. Chem., 1995, 270, 26723-26726

37) Atkin, C., Neilands, J., Rhodotorulic acid, a diketopiperazine dihydroxamic acid with growth-factor activity. I. Isolation and characterization. Biochemistry, 1986, 7, 3734–3739

38) Ankenbauer, R., Toyokuni, T., Staley, A., Rinehart, K., Cox, C., Synthesis and biological activity of pyochelin, a siderophore of Pseudomonas aeruginosa. J Bacteriol. 1988, 170, 5344–5351

16

CHAPTER TWO MELITTIN ANALOGUES

Melittin

Melittin is a hemolytic AMP isolated from the European honey bee Apis

mellifera. Melittin is the primary toxic component in bee venom and accounts for

approximately 50% of the dry mass. Melittin, figure 2.1, is a short linear peptide

containing 26 amino acids (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2). The residues

at the N-terminus are mostly hydrophobic while the residues at the C-terminus are mainly

hydrophilic due to the basic amino acids. The properties of melittin have promoted its use

as a model peptide for hemolytic peptides.

Figure 2.1: The structure of melittin.

Melittin, as with other cytolytic peptides, is mainly hydrophobic, however the

peptide has a net charge of +6 at physiological pH. Most of this charge, +4, is located on

the highly basic C-terminus containing Lys-Arg-Lys-Arg. Figure 2.2 shows that most of

the positive charge resides on one side of the peptide helix which provides the

am

h

b

h

in

F

ce

ag

an

p

mphiphilic c

ighly import

etween the p

ead groups o

nteractions b

igure 2.2: H

In aqu

ertain condit

ggregate into

nd high salt

eptide and a

character alo

tant for the m

peptide and t

of the memb

become weak

Helical wheel

ueous solutio

tions the pep

o tetramers.

content.2 Th

allows for the

ong with othe

mechanism o

the membran

brane; withou

ker.

l diagram of

on, melittin e

ptide can be

These condi

hese conditio

e formation

er hydrophil

of action.1 A

ne are betwe

ut the cation

f melittin.

exists as a m

induced to f

itions includ

ons negate th

of aggregate

lic groups. T

As stated befo

een the catio

nic charges in

monomeric ra

form a helica

de high pH, h

he charge rep

es. In the hel

The charged C

ore, the initi

nic residues

n melittin, th

andom coil.2

al secondary

high melittin

pulsion betw

lical conform

C-terminus i

al interaction

and anionic

hese and the

2 However, u

y structure an

n concentrati

ween melittin

mation, melit

17

is

ns

c

under

nd

ions,

n

ttin

co

sh

F

ac

qu

is

is

co

h

as

m

ontains two

hape, figure

igure 2.3: 3D

Melittin’

ctivity is me

uickly to ent

s approximat

s attributed t

ontains 1.8x

owever evid

In additio

s well. Melit

melittin’s min

α-helical sub

2.3.

D representa

s primary ta

elittin’s distin

throcytes. Th

tely 1-2 µM

o the large a

x107 binding

dence suppor

on to hemoly

ttin is active

nimum inhib

bunits separa

ation of meli

Ac

arget is the en

nguishing ch

he melittin c

depending o

amount of bi

sites.4 The b

rts the torida

ytic activity,

against gram

bitory concen

ated by a pro

ittin showing

ctivity of M

nthrocyte me

haracteristic.

concentration

on experime

nding sites f

binding mec

al pore mode

, melittin pos

m-positive a

ntration (MI

oline-14 resi

g the “bent r

elittin

embrane and

.3 At lower c

n needed to

ental conditio

for melittin.

hanism has b

el described

ssesses a str

and gram-neg

IC) against S

idue to give

rod”.

d, as such, th

concentration

cause 50% h

ons.3 The low

For instance

been though

in chapter 1

rong antibact

gative bacter

S. Aureus is 3

a “bent-rod”

he hemolytic

ns melittin b

hemolysis (H

w concentrat

e, an enthroc

h much deba

.5,6

terial compo

ria. For exam

3.6 µM and

18

”

c

binds

HC50)

tion

cyte

ate,

onent

mple,

19

against E. Coli, 3.9 μM.7 The exploitation of this low MIC and the reduction of hemolytic

activity has been the focus of much research.

Structural Requirements for Activity Several extensive studies of melittin have determined that the hemolytic and

antibacterial activities are independent of one another. In the early structural activity

relationship studies in to the requirements of melittin it was determined the N-terminal 1-

20 residues play mainly a structural role in melittin’s absolute activity.8 Additionally, it is

determined that the C-terminal 21-26 residues are a necessary component for hemolytic

activity.8 However, more recent studies have proven that this is not the case.

In a comprehensive study by Blondelle and Houghten, the necessary residues

were discovered for melittin’s hemolytic and antibacterial properties. The individual

omission of Leu-6, Leu-9, Leu-13, or Leu-16 give an over 100-fold decrease in hemolytic

activity.9 This is also evidence to support the need for the leucine zipper design for

aggregation in the plasma membrane. The omission of Ile-17 and Trp-19 also leads to

considerable decrease in hemolytic activity. However, the omission of the previously

described amino acids did not have the same drastic effects on the antibacterial activity.

Only a 2-fold or 3-fold decrease in antibacterial activity was observed in contrast to the

100-fold seen in the hemolytic studies.

Blondelle and Houghten also explored an individual leucine substitution of each

amino acid in the melittin peptide. It is interesting to note that at 19 amino acids that were

substituted had considerably higher hemolysis percentage than the unadulterated

20

melittin.10 Two amino acids of note, Lys-7 and Trp-19 had detrimental effects on the

hemolytic activity when substituted, even at the highest concentrations tested.10

Unfortunately, the antibacterial activity was not probed with these analogues.

A core requirement for hemolytic activity is the amphiphilic helical secondary

structure, yet the helical structure is not a requirement for antibacterial activity. In a study

by Oren and Shai, four amino acid residues, Val-5, Val-8, Iso-17 and Lys-21, were

replaced with D-isomers of the respective amino acids. The melittin analogue showed a

low propensity to form a helical secondary structure. Because the peptide had low

propensity for helix formation, the hemolytic activity was also low, yet there was still a

significant level of antibacterial activity.11

Truncated Melittin

As described previously, it is documented that the N-terminus plays a minimal

role with the mechanism for antibacterial activity. As such, researchers have explored

using truncated melittin fragments to determine their viability as therapeutics. Extensive

studies have focused on the fragment around residues 12-25, figure 2.4, containing the C-

terminal basic amino acids.12-14 The peptide has a lower propensity to form a helical

secondary structure which makes it an ideal lead compound to study. As a result, the

hemolytic activity is reduced while the antibacterial activity is retained.

21

Figure 2.4: The structure of truncated melittin(12-25).

1st Genertation Melittin Derivatives

The current project explored the coupling of melittin, metal destabilization, and

siderophores as targeted antimicrobial peptides.. Previous studies have shown that Thr-

10 and Lys-21 are not critical for antibacterial activity, so, in the present project, these

sites were selected for incorporation of ligands for metal binding. Thr-10 was changed to

Lys-10 to be able to couple the ligand to the peptide at residues 10 and 21. Two ligands

were selected to determine the possible differences between bidentate ligand and

tridentate ligands.

Figure 2.5: Placement of metal binding moieties to melittin, where X represents bipyridine or terpyridine.

The design of this work was to use metal chelation to deactivate the peptide’s

hemolytic activity and retaining antibacterial activity with a metal-ligand switch. The

targeted activity will activate once a siderophore from a bacteria will remove the metal

ion and reactivate the peptide. Since siderophores are released from bacteria must remain

in

p

p

re

F

n close proxi

eptide to atta

eptide will b

eleased.

igure 2.6: M

imity to be a

ack the bacte

be deactivate

Metal assisted

absorbed bac

eria instead

ed with a me

d deactivatio

ck into a bac

of healthy ce

etal ion, and

on of peptide

cterium.15 Th

ells. Figure 2

then reactiv

e with sidero

he proximity

2.6 represen

vated when th

ophore reacti

y will allow t

nts how the

he metal ion

ivation.

22

the

n is

23

Synthesis of Metal Binding Moieties

Scheme 2.1: Synthesis of metal binding ligands.

To preserve the positive charge required for melittin activity, two neutral metal

binding ligands were chosen as chelators, 4-methyl-2,2'-bipyridine-4'-carboxylic acid

and 2,2’:6’, 2”-terpyridine-4’-carboxylic acid, scheme 2.1. The carboxylic acid moieties

will be used to couple the ligand to a lysine residue with the modified melittin. The

compounds were synthesized according to literature procedures. To synthesize the

bipyridyl compound 3, commercially available compound 1 was selectively oxidized

with selenium dioxide. Compound 2 was further oxidized with silver oxide to afford the

carboxylic acid in compound 3.16 The terpyridyl compound 6 was synthesized by treating

commercially available citrazinic acid (4) with phosphorous oxybromide to produce

compound 5.17 The dibromide compound 5 was then used in a Stille coupling reaction

using a palladium catalyst and 2-(tributylstannyl)pyridine to provide the desired final

product, compound 6.18 The methyl ester is hydrolyzed during the work up procedure.

24

Synthesis of Melittin Derivatives

Scheme 2.2: Synthesis of melittin derivatives, where X is terpyridine or bipyridine.

The melittin derivatives MLT-bpy and MLT-tpy, figure 2.5, were synthesized by

solid phase peptide synthesis following the Fmoc based protocol. 4-methyltrityl (Mtt)

protected lysine was used to selectively allow binding of the ligand to the desired lysine

residue. Other lysine residues were protected with the allyloxycarbonyl (alloc) protecting

group. The solid support for the synthesis was the ChemMatrix rink amide resin with a

0.52 mmol/g loading. The coupling reactions were monitored using the ninhydrin test

(Kaiser test) for primary amines and the chloranil test for secondary amines. Once the

peptide was completed, the Mtt protecting groups were removed with 1.8%

trifluoroacetic acid in methylene chloride. The reaction mixture was drained and a yellow

solution indicated the removal of the protecting group. More deprotection cocktail was

added and repeated until little to no color appeared in the reaction mixture. The ligand

compounds were coupled to the free amine of the deprotected lysine using standard

25

HBTU coupling. The remaining Alloc protecting groups were deprotected using a

palladium catalyzed decoupling procedure. The peptides were cleaved from the resin with

non-acid liable protecting groups removed with a cleavage cocktail composed of 95%

TFA, 2.5% TIPS, 2.5% water. The peptides were then purified by reverse phase HPLC

and characterized Matrix-assisted laser desorption ionization (MALDI) mass

spectroscopy, table 2.1.

Table 2.1: Purification of melittin derivatives.

Compound HPLC

%acetonitrile/waterRetention

Time Actual Mass

Observed Mass

MLT-bpy 35-70 25 min 3308.04 3308.99

MLT-tpy 45-80 27 min 3434.09 3435.56

Circular Dichroism

Circular dichroism experiments were conducted to determine helicity of the

designed peptides, figure 2.7. In 100mM phospate buffer at pH 7.4 showed that the

peptide was found to form an α-helix. Interestingly, when iron (II) as added to the

peptides in solution, little change was observed in the helicity of MLT-bpy, but the

helical characteristics were somewhat lost with MLT-tpy.

F

7

F7

igure 2.7: C

.4.

igure 2.8: C.4, with 1 eq

D spectra of

D spectra ofquivalent Fe(

f melittin der

f melittin der(II).

rivatives, 30

rivatives, 30

0 μM, in 100

0 μM, in 100

0 mM phospa

0 mM phospa

ate buffer, pH

ate buffer, pH

26

H

H

n

4

U

un

ac

F

se

The an

egative bact

0° C for 6 ho

Unfortunately

nmodified m

ctivity.

igure 2.9: E.

The n

elected as it

ntimicrobial

erium, Esch

ours and the

y, the peptid

melittin. Ther

. coli antibac

ext peptide d

possesses lo

Ant

l activity of t

erichia coli.

e optical dens

des exhibited

refore, a new

cterial assay

2nd Genera

design began

ower hemoly

timicrobial A

the peptides

19 The pepti

sity was mea

d poor antimi

w design was

y of melittin d

ation Melitti

n with a trun

ytic activity a

Activity

were explor

ides were inc

asured to de

icrobial activ

s developed

derivatives.

in Derivative

ncated melitt

as compared

red initially w

cubated with

termine viab

vity as comp

to increase

6 hour incub

es

tin. This pep

d to melittin,

with the gra

h the bacteria

bility.

pared to

antimicrobia

bation.

ptide was

but retains

27

am-

a at

al

28

similar antibacterial activity. The metal ligands were also moved to the terminus of the

peptide in an effort to have less effect on activity. Four truncated peptides, figure 2.10;

containing melittin residues 12-25 with metal binding ligands were synthesized to

determine their antibacterial activity with and without metal ions. Two of the peptides

contained a single ligand on each of the C- and N- termini of the peptides as shown

below. The third peptide contained ligands at both termini. We also prepared a truncated

12-25 residue melittin fragment as a control peptide. The same synthetic strategy was

used to attach the ligand to the lysine residues. A glycine spacer was between the

truncated peptide and the ligand-modified lysine residues at the termini.

Figure 2.10: Second generation melittin derivatives. From top to bottom: MLTx, MLTx1, MLTx2, MLTx3. Where X is terpyridine.

Synthesis of 2nd Generation Melittin Derivatives

The new peptides were synthesized in the same manner as described previously.

Terpyridine and bipyridine were used as ligands, however, the coupling of of the

bipridine MLTx1 proved to be difficult and was abandoned, nevertheless, MLTx2-bpy

29

was completed. Given that bipyridine is a bidentate ligand and would most likely bridge

multiple peptides leading to higher ordered structures, only terpyridine was used in the

synthesis of MLTx3. The peptides were then purified by reverse phase HPLC and

characterized Matrix-assisted laser desorption ionization (MALDI) mass spectroscopy,

table 2.2.

Table 2.2: Purification of truncated melittin derivatives.

Compound HPLC

%acetonitrile/waterRetention

Time Actual Mass

Observed Mass

MLTx 10-50 38 min 1887.28 1886.45 MLTx1-tpy 10-40 48 min 2109.52 2110.01 MLTx2-tpy 20-50 30 min 2110.98 2109.96 MLTx3-tpy 20-60 26 min 2554.01 2553.54

UV/Vis spectroscopy

UV/Vis spectroscopy was used to monitor that the ligands within the peptide are

chelating the metal. There is a distinctive charge transfer that occurs between terpyridine

and iron(II), with the λmax for the terpyridine-iron(II) complex occurring at around

570nm. The spectra below show that the charge transfer is occurring, demonstrating that

the peptides do bind to the metal ions.

F

F

h

fi

α

igure 2.11: U

igure 2.12: U

Circul

elicity of the

igure 2.10, a

-helix in 10m

UV/Vis spec

UV/Vis spec

lar dichroism

e peptides. T

and 10% hex

mM phospha

ctroscopy of

ctroscopy of

Ci

m experimen

The peptides

xafluoroisopr

ate buffer an

f MLTx1-tpy

f MLTx3-tpy

ircular Dichr

nts were cond

were tested

ropanol (HFI

nd not surpri

y:Fe(II), 100

y:Fe(II), 100

hroism

ducted on ea

in 10 mM p

IP)/water, fi

singly has a

0 μM, in wate

0 μM, in wate

ach peptide t

phosphate bu

igure 2.13. M

higher degr

er.

er.

to determine

uffer at pH 7

Melittin form

ree of helicit

30

e

7.4,

ms an

ty as

31

it has a longer sequence. The truncated melittin fragments did not possess appreciable

helicity in the 10 mM phosphate buffer, but, unexpectedly MLTx3-tpy did possess

moderate helicity in 10 mM phosphate buffer. Furthermore, all peptides showed

appreciable helicity in 10% HFIP, figure 2.14. Hexafluoroisopropanol is commonly used

to induce helicity of peptides which form α- helical secondary structures.

When metal ions were added to the peptides, figure 2.14, it was observed that the

helix is destabilized in solution. Interestingly, the denatured peptide appears to retain

more helical characteristics. The peptide was denatured in 10 mM urea solution, then iron

(II) was added to solution. The urea was removed from solution by dialysis filtration. The

denaturing of the peptide was necessary because of the possibility that the peptide may be

partially formed into a helix. This might contribute to a higher than normal antibacterial

activity.

F

F

igure 2.13: C

igure 2.14: C

CD of melitt

CD of melitt

tin derivative

tin derivative

es, 50 μM, in

es, 50 μM, in

n 10 mM ph

n 10% HFIP

hosphate buf

P/water

ffer, pH 7.4

32

Fbu

co

fo

M

th

b

th

to

1

p

d

igure 2.15: Cuffer, pH 7.4

The an

oli and Gram

or 6 hours an

MLTx were u

he same antib

etween the r

he reported p

o continue ov

2-25 is 4 µM

eptides show

erivatives. A

CD of melitt4. MLTx3d-

ntimicrobial

m-positive S.

nd the optica

used as the c

bacterial act

reported and

procedure by

vernight, but

M, nearly ide

wed consider

Adding one e

tin derivativetpy was den

Ant

l activity of t

. aureus. The

al density wa

control peptid

tivity as was

d the present

y adding sod

t the results

entical to the

rably more a

equivalent o

es, 50 μM, wnatured prior

timicrobial A

the peptides

e peptide wa

as measured

des. Howeve

reported.19

project's pro

dium chloride

were not con

e full melittin

activity than

of FeCl2 to th

with metal ior to metal add

Activity

was explore

as incubated

to determin

er, MLTx be

This is perh

ocedures. At

e, 5 grams/li

nsistent.19 Th

n sequence. I

MLTx and t

he peptide di

on in 10 mMdition.

ed using Gra

d with the ba

ne cell viabili

ecause MLT

aps due to th

ttempts were

iter, and allo

he reported

Interestingly

the 1st-gener

id not deacti

M phosphate

am-negative

cteria at 40°

ity. Melittin

Tx did not sh

he difference

e made to mi

owing the as

MIC for me

y, the modifi

ration

ivate either

33

E.

° C

and

how

es

imic

say

littin

ied

34

MLTx1 or MLTx2 containing the terpyridine ligand. However, some decrease in

antibiotic activity did occur with MLTx3-tpy. A denatured peptide, MLTx3d-tpy, was

also tested. The peptide was first denatured in 10mM urea, then the metal ion was added

to solution. The solution was than dialyzed to remove the urea from the solution. By

denaturing the peptide, the activity of the peptide-metal complex (MLTx3d: Fe(II)) was

lowered 2-fold as compated to the non-denatured sample (MLTx3: Fe(II)).

Table 2.3: MIC of melittin derivatives.

Peptide E.Coli S. Aureus

Melittin 4 μM ± 0.002 1 μM ± 0.006

MLTx >64 μM ± 0.02 -

MLTx1-tpy 16 μM ± 0.001 8 µM ± 0.019

MLTx2-tpy 16 μM ± 0.02 8 µM ± 0.02

MLTx3-tpy 8 μM ± 0.012 8 µM ± 0.009

MLTx1-tpy: Fe(II) 16 μM ± 0.08 8 μM ± 0.02

MLT2x-tpy: Fe(II) 16 μM ± 0.02 8 μM ± 0.02

MLTx3-tpy: Fe(II) 16 μM ± 0.006 8 μM ± 0.01

MLTx3d-tpy: Fe(II) 32 μM ±0.020 16 μM ± 0.02

Reversibility of the Metal-Ligand Interaction

Terpyridine-Fe(II) interactions are known to be reversible in polymers and

peptides.21,22 However, attempts to remove the metal ion from the melittin derivatives has

proven to be very difficult. Several solvents and chelators were used to attempt the

removal of the metal ions, figure 2.16. Water, methanol, and dimethylformamide (DMF)

w

an

T

w

FF

on

M

U

o

C

sh

were the solv

nd N-(hydro

The solutions

was only brok

igure 2.16: Me(II) comple

Other

nly cobalt af

MLTx3-tpy w

UV/Vis spect

ccurred. Th

Co(II) to the p

howed little

vents used, w

oxyethyl)-eth

s were heated

ken with the

MLTx3-tpy ex with 2 equ

possible for

fforded a me

was analyzed

tra of MLTx

he CD spectr

peptide dest

difference in

while deferox

hylenediamin

d to 50° C an

e peptides in

with 1 equivuivalents of

Melittin De

r metal ions

etal-ligand ch

d with 1 equi

x3-tpy: Co(II

ra of MLTx3

abilized the

n the spectru

xamine, ethy

netriacetic a

nd kept for 1

DMF as can

v Fe(II) in sochelators.

rivatives wit

were explor

harge transfe

ivalent of m

I), in shows t

3-tpy: Co(II)

helix, and d

um.

ylenediamine

acid (HEDTA

18 hours. Th

n been seen b

olution. 500

th Other Me

red: cobalt, c

fer that was o

metal ion as p

the metal lig

, figure 2.18

denaturing th

etetraacetic a

A) were the c

he metal-liga

by the comp

µM solution

etals

copper, and n

observable, f

previously de

gand charge

8, shows that

he peptide wi

acid (EDTA

chelators use

and interactio

plete color lo

n of the pept

nickel. How

figure 2.17.

escribed.The

transfer that

t adding the

ith a 10 mM

35

A),

ed.

on

oss.

tide-

ever,

e

t

M urea

Fco

F

igure 2.17: Uontrol (red).

igure 2.18: C

UV/Vis spec

CD of melitt

ctrum of ML

tin derivative

LTx3-tpy:Co

es, 50 μM, in

o(II), 100 μM

n 10 mM ph

M, (blue) and

hosphate buf

d terpyridine

ffer, pH 7.4.

36

e

37

Conclusions

Melittin derivatives are susceptible to small changes which have major impact to

their activity. The 1st-generation peptides ultimately failed to maintain any antibacterial

activity. The addition of the aromatic ligands at residues 10 and 21 was enough to reduce

the antibacterial activity 30-fold. However, the truncated peptides show promise as novel

antibacterials as they have retained most of their activity. Unfortunately, the difficulty of

reversing the metal-ligand interaction is a problem that needs to be solved to move the

project further.

Materials and Methods

All peptides were synthesized on Rink amide resin (200-400 mesh) purchased

from novabiochem. Critizanic acid and 4,4'-dimethyl-2,2' bipyridine were both

purchased from VWR International. Other reagents used were purchased from Sigma-

Aldrich or VWR International. The peptides were purified and analyzed with Waters

Delta Prep 4000 HPLC. The ligand intermediates and final compounds were

characterized by NMR spectroscopy with the Varian Inova-300 Mhz nuclear magnetic

resonance (NMR) spectrometer. All peptides were characterized by mass spectrocopy

using an Applied Biosystems Voyager-DE Matrix-assisted laser desorption ionization –

time of flight mass spectromerter. Circular dichroism experiments were performed on a

Jasco J-810 circular dichroism spectrophotometer. UV/Vis spectra were obtained on a

Cary 100 Bio UV-visible spectrophotometer.

38

Experimental

4-methyl-2,2'-bipyridine-4'-carboxaldehyde(2) To a dry 500 ml round bottom flask

was added 4,4'-dimethyl-2,2' bipyridine (1) (5.00 g, 27 mmol) followed by 200 ml

dioxane. The solution was sparged with nitrogen for 15 minutes with stirring. Selenium

dioxide (3.31 g, 30 mmol) was then added and the solution was sparged while stirring for

15 minutes. The solution was heated to a gentle reflux for 24 hours. After cooling the

reaction mixture was vacuum filtered and the solvent removed. The remaining solid was

dissolved in 100 ml ethyl acetate and refluxed for an additional 1 hour and vacuum

filtered while hot. The filtrate was washed with 0.1 M sodium carbonate followed by 0.3

M sodium metabisulfate. The pH was then adjusted to 10 with solid sodium carbonate

and then extracted with methylene chloride. (yield: 1.67 g, 31%) 1H-NMR (300 MHz,

CD2Cl2) δ 10.19 (s, 1H), 8.84(d, 1H), 8.80(s, 1H), 8.52(d, 1H), 8.29(s, 1H), 7.71(d, 1H),

7.22(d, 1H), 2.47(s, 3H)

4-methyl-2,2'-bipyridine-4'-carboxylic acid(3) To a dry 250 ml round bottom flask

was added 95% ethanol/water solution followed by 4-methyl-2,2'-bipyridine-4'-

carboxaldehyde (2) (1.67 g, 8.40 mmol). An aqueous 20 ml solution of silver nitrate (

1.56 g, 9.20 mmol) was then added. A 1 M solution of sodium hydroxide (1.67 g, 42

mmol) was added via an addition funnel for 20 minutes and the reaction mixture was

stirred for 24 hours. The ethanol was removed by reduced pressure. The reaction mixture

was then vacuum filtered and the solids washed with 2 M sodium hydroxide followed by

water. The filtrate was extracted with methylene chloride. The pH of the aqueous solution

was adjusted to 3. The lower pH allowed a white precipitate to form and was collected.

39

(yield: 0.746g, 41%) 1H -NMR (300 MHz, (CD3)2SO) δ 8.86(d, 1H), 8.81(s, 1H), 8.57(d,

1H), 8.26(s, 1H), 7.86(d, 1H), 7.33(d, 1H), 2.48(s, 3H)

2,6-Dibromoisonicotonic acid methyl ester (5) Phosphorous oxybromide (10 g, 35

mmol) and citrazinic acid (4) (1.7 g, 11 mmol) were ground together, put in a dry 100 ml

round bottom flask and purged with nitrogen. The reaction mixture was heated to 180

ºC under an nitrogen atmosphere for 90 min. The mixture was then cooled to room

temperature and quenched with anhydrous MeOH (10 mL). After dilution with saturated

aqueous K2CO3 (20 ml), the solution was extracted with dichloromethane (3x20 ml). The

organic layer was washed with brine and dried over sodium sulfate. The solution was

decanted into a 1 inch silica plug and washed with DCM (3x15 ml). The filtrate solution

was evaporated under lower pressure. No further purification was necessary. (yield: 2.22

g, 68.5%) 1H -NMR (300 MHz, CD2Cl) δ 8.00(s, 2H), 3.97(s, 3H)

2,2':6', 2"-terpyridine-4'-carboxylic acid (6) 2,6-Dibromoisonicotonic acid methyl

ester (1.6 g, 5.4 mmol), tributylstannyl pyridine (3.51 ml, 10.8 mmol), and

tetrakis(triphenylphosphine)palladium (0.63 g, 0.54 mmol) were dissolved in 50 ml dry

toluene in a dry 150 ml round bottom flask. The reaction mixture was heated to 110° C

under nitrogen for 16 hours. The reaction was monitored by TLC. Once the reaction was

complete, the mixture was cooled to room temperature and quenched with saturated

ammonium chloride (30 ml) and stirred for additional 30 minutes. The reaction mixture

was filtered over celite and the filtered solids washed with DCM. The aqueous phase was

extracted with DCM (3x15 ml). The organic phases were dried with sodium sulfate and

the solvent removed. Concentrated hydrochloric acid (15 ml) was added to the residue to

hydrolyze the methyl ester. The mixture was neutralized with 2 M sodium hydroxide and

40

extracted with DCM (3x10 ml). The organic layer was separated and the solvent

evaporated. A 2/1 water/methanol mixture (25 ml) was added to the residue. 2 M sodium

hydroxide solution (15 ml) was added and the mixture was stirred for 1 hour, this was to

complete the hydrolysis of the methyl ester. The solution was extracted with DCM. The

aqueous layer was then acidified to pH 3 and then the methanol removed under reduced

pressure. The pH was adjusted to 6 which allowed a white precipitate to form. (yield:

0.983 g, 65.6%) 1H -NMR (300 MHz, CD3OD) δ 8.86(s, 2H), 8.71(m, 4H), 8.02(td, 2H),

7.49(qd, 2H)

General Synthesis of Peptides. In a 20 ml peptide synthesis flask was added 200 mg

Rink-Amide resin (0.052 mmol/g). The peptide was shaken with 10ml DCM to swell the

resin. Fmoc-protected amino acids (6 equiv), HBTU (6 equiv), DIEA (12 equiv) were

dissolved in 10 ml DMF and added to the resin. The synthesis flask was spun for 4 hours.

The resin was washed with DMF, methanol, and DCM (3x10 ml). Piperidine (25% in

DMF, 10 ml) was added to the resin and spun for 20 minutes and then drained. The

peptide was washed with DMF, methanol, and DCM (3x10 ml). The procedure was

continued until the peptide was complete.

Kaiser Protocol Approximately 20 resin beads were removed from the synthesis flask

and placed in a small culture tube. Three drops of ninhydrin solution (1.0 g of ninhydrin

in 20 mL of n-butanol), three drops of phenol solution (40 g of phenol in 20 mL of n-

butanol), and three drops of KCN solution (dissolve 16.5 mg of KCN in 25 mL of

distilled water. Dilute 1.0 mL of KCNaq solution with 49 mL of pyridine) were added to

the culture tube. The mixture was added to a beaker of boiling water. If the beads turned

41

blue there is a free primary amine. If there is no color change or if the beads turn slightly

brown there are not any free primary amines present.

Chloranil Protocol Approximately 20 resin beads were removed from the synthesis flask

and placed in a small culture tube. Three drops of chloranil solution (2% chloranil in

DMF) and three drops of acetylaldehyde solution (2% acetylaldehyde in DMF) were

added to the culture tube. The mixture was allowed to sit at room temperature. A blue-

green beads indicates a free secondary amine. A clear resin indicated were not any free

secondary amines present.

4-Methyltrityl (Mtt) deprotection The resin was washed with DCM then 15 ml of 1.8%

TFA in chloroform and then added to the resin. The resin was allowed to stir for 4

minutes. The yellow solution was drained and the resin washed with DCM. The

procedure was continued until little yellow color evolved from the solution.

Coupling of metal-chelating ligand The resin was washed with DMF. The ligand (6

equiv), HBTU (6 equiv), DIEA (12 equiv) were dissolved in 10 ml DMF and added to the

resin. The resin was spun for 4 hours then washed with DMF, methanol, and DCM (3x10

ml). The resin was tested to ensure complete coupling. If a positive Kaiser test occurred,

a second coupling procedure was performed.

Aloc deprotection The resin was washed with chloroform and an aloc cleavage cocktail

was added to the resin in 15 ml chloroform/acetic acid (0.5 mL per gram of resin), N-

methylmorpholine (2 mL per gram of resin), and Pd(PPh3)4 (0.3 equivalents based on

resin substitution). The resin was spun for 45 minutes and analyzed by MALDI. If the

reaction was not complete, the procedure was repeated if necessary.

42

CD Protocol A peptide sample was taken from the stock solution and diluted in 10 mM

phosphate buffer solution pH 7.4 or 10% HFIP in water to make a 200 µL solution. The

sample solution was scanned from 260 nm to 190 nm using a 1 nm bandwidth.

UV/Vis Protocol A peptide sample was taken from the stock solution and diluted with DI

water to a volume of 500 µL. 1 µL of a stock FeCl2 solution was added to the peptide

solution and a color change was observed. The solution was then scanned.

Denaturation Protocol A sample of stock peptide solution was diluted in a 10 mM urea

solution. After 15 min 1 equivalent of a stock FeCl2 solution was added to the solution

and a color change observed. The solution was then added to a dialysis tubing. The

tubing was placed in a 1 L beaker containing DI water. The solvent was changed over 30

minute intervals twice. The solvent was changed again and left overnight.

Antibacterial Assay Procedure Bacteria were grown on a TSB agar culture plate

overnight at 40° C. The bacteria were inoculated in 50 ml TSB media until the OD600 was

between 0.4 and 0.6. 5 ml of the culture media was centrifuged and washed with MHB

media (3x 5 ml). The culture media was diluted to an OD600 of 0.001. 90 µl of the

bacterial solution was pipetted on a 96 well plate along with 10 µl peptide solution. The

96 well plate was incubated at 40° C with shaking for 6 hours. The OD600 was taken and

the lowest concentration where there is minimal growth was decided to be the MIC.

43

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