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January 2015

Chemical Synthesis of Bacterial Siderophores andApplications in Pathogen DetectionGregory Gene JarvisPurdue University

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Recommended CitationJarvis, Gregory Gene, "Chemical Synthesis of Bacterial Siderophores and Applications in Pathogen Detection" (2015). Open AccessTheses. 1110.https://docs.lib.purdue.edu/open_access_theses/1110

PURDUE UNIVERSITY GRADUATE SCHOOL

Thesis/Dissertation Acceptance

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.

Gregory G. Jarvis

CHEMICAL SYNTHESIS OF BACTERIAL SIDEROPHORES AND APPLICATIONSIN PATHOGEN DETECTION

Master of Science

Philip S. Low

Christine A. Hrycyna

David Thompson

Philip S. Low

R. E. Wild 03/09/2015

i

CHEMICAL SYNTHESIS OF BACTERIAL SIDEROPHORES AND APPLICATIONS

IN PATHOGEN DETECTION

A Thesis

Submitted to the Faculty

of

Purdue University

by

Gregory G. Jarvis

In Partial Fulfillment of the

Requirements for the Degree

of

Master of Science

May 2015

Purdue University

West Lafayette, Indiana

ii

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank all of my family, friends, and

colleagues who have supported me throughout my time studying chemistry at Purdue. I

sincerely appreciate the time that post doctoral researchers Rajesh Pandey, Madduri

Srinivasarao, and Srinivasa Tenneti have willingly dedicated to mentoring me during my

time at Purdue, allowing me thrive as an independent researcher. I especially want to

thank my parents, Gary and Terri Jarvis, who have always been there to provide sound

wisdom and guidance through the arduous times that accompany graduate school. They

have always encouraged me to follow my own path, allowing me to become the

individual that I am today.

iii

TABLE OF CONTENTS

Page

LIST OF FIGURES ............................................................................................................ v 

LIST OF SCHEMES.......................................................................................................... vi 

LIST OF ABBREVIATIONS ........................................................................................... vii 

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

CHAPTER 1. AN IMPROVED SYNTHESIS OF PETROBACTIN, A BACILLUS ANTHRACIS SIDEROPHORE……..…………………………………………........................1

1.1  Abstract ........................................................................................................ 1 1.2 Introduction .................................................................................................. 1 1.3 Experimental Section ................................................................................... 3 1.3.1 Chemicals and Materials .......................................................................... 3 1.3.2 Equipment ................................................................................................. 3 1.3.3 Synthesis of 3-(dibenzylamino)propyl methanesulfonate (3) .................. 3 1.3.4 Synthesis of tert-Butyl (4-((3- (dibenzylamino)propyl)amino)butyl)carbamate (5) .................................. 4 1.3.5 Synthesis of tert-butyl (4-((3-aminopropyl)amino)butyl)carbamate (6).. 4 1.3.6 Synthesis of methyl 3,4-bis(benzyloxy)benzoate (9) .............................. 4 1.3.7 Synthesis of tert-Butyl (4-((3-(3,4-

bis(benzyloxy)benzamido)propyl)amino)butyl)carbamate (10) .............. 5 1.3.8 Synthesis of N-(3-((4-aminobutyl)amino)propyl)-3, 4-bis(benzyloxy)benzamide (11) ................................................................ 5 1.3.9 Synthesis of 2-tert-Butyl-1,3-di-N-(hydroxyl)-Succinimidyl Citrate (13) ................................................................................................ 6 1.3.10 Synthesis of tert-butyl 4-((4-((3-(3,4- bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-(2-((4-((3-(3,4- bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-oxoethyl)-2- hydroxy-4-oxobutanoate (14) .................................................................. 6 1.3.11 Synthesis of Petrobactin (1) ...................................................................... 6 1.4 Results and Discussion ................................................................................. 7 1.5 Conclusion .................................................................................................... 8

iv

Page CHAPTER 2. CHEMICAL SYNTHESIS OF STAPHYLOFERRIN A AND ITS EVALUATION FOR USE IN A PATHOGEN-DETECTION DEVICE…………….…….10

2.1  Abstract ...................................................................................................... 10 2.2 Introduction ................................................................................................ 10 2.3 Experimental Section ................................................................................. 12 2.3.1 Chemicals and Materials ........................................................................ 12 2.3.2 Equipment ............................................................................................... 13 2.3.3 Synthesis of Staphyloferrin A ................................................................ 13 2.3.4 Preparation of Staphyloferrin A-BSA Conjugate ................................... 15 2.3.5 Preparation of Staphyloferrin A-BSA-patterned Chips .......................... 15 2.3.6 Growth of S. aureus ................................................................................ 16 2.3.7 Capture and Imaging of Chips ............................................................... 17 2.4 Results and Discussion ............................................................................... 17 2.5 Conclusion .................................................................................................. 18

CHAPTER 3. SELECTIVE CAPTURE OF S. AUREUS USING IMMOBILIZED SIDEROPHORE STAPHYLOFERRIN A…………………………………………………...21

3.1  Abstract ...................................................................................................... 21 3.2 Introduction ................................................................................................ 21 3.3 Experimental Section ................................................................................. 23 3.3.1 Chemicals and Materials ........................................................................ 23 3.3.2 Equipment ............................................................................................... 23 3.3.3 Synthesis of Staphyloferrin A ................................................................ 24 3.3.4 Preparation of Staphyloferrin A-BSA conjugate .................................... 25 3.3.5 Preparation of Staphyloferrin A-BSA-patterned chips ........................... 25 3.3.6 Growth of S. aureus ................................................................................ 25 3.3.7 Capture and Imaging of Chips ............................................................... 26 3.3.7.1 Evaluation of Limit of Detection ......................................................... 26 3.3.7.2 Time-course Study ............................................................................... 26 3.3.7.3 Evaluation of Chip Specificity ............................................................ 27 3.4 Results and Discussion ............................................................................... 27 3.5 Conclusion .................................................................................................. 29

LIST OF REFERENCES .................................................................................................. 35 

PUBLICATIONS .............................................................................................................. 41 

v

LIST OF FIGURES

Figure ............................................................................................................................. Page 1.1 Structure of petrobactin, 1 ............................................................................................ 8

2.1 Capture of S. aureus using a lab-on-a-chip device consisting of SA-BSA stamped on a gold substrate ............................................................................................................ 20

3.1 Chemical structure of ferric-staphyloferrin A with a short peptide linker for use in capture studies (15) ........................................................................................................... 30

3.2 The microcontact printing process used on gold chips to produce our lab-on-a-chip device. ............................................................................................................................... 32

3.3 Capture efficiency based on concentration of S. aureus ............................................. 33

3.4 Time-course study to examine the effect of bacterial exposure time to the chip ....... 33

3.5 Specificity of capture device when tested against a variety of human pathogens ...... 34

vi

LIST OF SCHEMES

Scheme Page

1.1 Chemical synthesis of the first building block of petrobactin ...................................... 8

1.2 Chemical synthesis of catechol subunit of petrobactin ................................................. 9

1.3 Chemical synthesis of functional petrobactin ............................................................... 9

2.1 Chemical synthesis of SA using SPPS techniques ..................................................... 19

3.1 Solid-phase synthesis of 15 ......................................................................................... 31

vii

LIST OF ABBREVIATIONS

PCR: Polymerase chain reaction

Sb(OEt)3: Antimony triethoxide

TEA: Triethylamine

DMSO: Dimethyl sulfoxide

EtOAc: Ethyl acetate

i-PrNH2: Isopropylamine

AcOH: Acetic acid

MRSA: Methicillin-resistant Staphylococcus aureus

VRSA: Vancomycin-resistant Staphylococcus aureus

SA: Staphyloferrin A

ABC transporter: ATP-binding cassette transporter

SPPS: Solid-phase peptide synthesis

3-t-Bu-citrate: 3-(tert-butoxycarbonyl)-3-hydroxypentanedioic acid

BSA: Bovine serum albumin

EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

PDMS: Polydimethylsiloxane

PBS: Phosphate-buffered saline

DCM: Dichloromethane

viii

THF: Tetrahydrofuran

DMF: N,N-Dimethylformamide

PyBOP: Benzotriazole-1yl-oxytripyrrolidinophosphonium hexafluorophosphate

DIPEA: N,N-Diisopropylethylamine

MeOH: Methanol

(Ph3P)4Pd0: Tetrakis(triphenylphosphine)palladium(0)

PhSiH3: Phenylsilane

TFA: Trifluoroacetic acid

TIPS: Triisopropylsilane

LoD: Limit of detection

ix

ABSTRACT

Jarvis, Gregory G. M.S., Purdue University, May 2015. Chemical Synthesis of Bacterial Siderophores and Applications in Pathogen Detection. Major Professor: Philip S. Low. Bacterial siderophores are a class of small organic compounds that are produced

endogenously by bacteria to chelate essential ferric iron from their surroundings. Because

the bioavailability of ferric iron is extremely low is physiological pH, the host of a

parasitic infection can be a poor source of soluble Fe3+. Consequently, bacteria rely upon

siderophore transport systems to efficiently scavenge the nutrient. Nearly every bacterial

species ever isolated has evolved a set of unique enzymatic pathways for de novo

synthesis of these compounds, thus exists an inimitable relationship between a genus or

species of bacteria and the siderophores it produces.

Our goal was to exploit the highly-refined interaction that takes place between a

siderophore and its corresponding membrane-bound transporter by utilizing it as means

to capture and identify a pathogen more rapidly and economically than standardized PCR

methodology. To achieve this, we developed a robust lab-on-a-chip device that consists

of a surface-mounted siderophore that is capable of capturing its cognate pathogen with a

high degree of accuracy. With this device, bacterial capture occurred in a predetermined

pattern, and was verified using a benchtop light microscope. This simple and relatively

inexpensive method of pathogen detection may allow for widespread use of the device,

while simultaneously reducing the time it takes to diagnose bacterial infections.

x

Until recently, researchers have obtained siderophores for research using

complicated, time-consuming techniques that involve isolating siderophore from large

bacterial supernatants followed by extensive purification. Our goal was to avoid

disadvantages associated with siderophore isolation methodology by chemically

synthesizing siderophores. This would allow us to circumvent the use of potentially

hazardous biologics while simultaneously improving product yield and drastically

simplifying the purification process. The following work describes an improved approach

for chemical synthesis of petrobactin, a Bacillus anthracis siderophore, as well as the first

chemical synthesis of staphyloferrin A (SA), a siderophore of Staphylococcus aureus. We

also outline the implementation of SA in an S. aureus detection device and report on its

efficacy.

1

CHAPTER 1. AN IMPROVED SYNTHESIS OF PETROBACTIN, A BACILLUS ANTHRACIS SIDEROPHORE

1.1 Abstract

A B. anthracis siderophore, petrobactin, was synthesized chemically with 65%

total yield. The synthesis includes application of a novel amide-ester exchange reaction

using Sb(OEt)3 under neat conditions, which considerably improved the chemical yield of

functional petrobactin when compared to other reported syntheses.

1.2 Introduction

Petrobactin, 1, is an iron-chelating siderophore produced by pathogenic bacterium

Bacillus anthracis (see figure 1.1).1 It has been shown to be a high-affinity scavenger of

sparingly-soluble ferric iron from the extracellular environment of a B. anthracis

infection.2-4 Since human pathogens require iron as an essential nutrient, the

reinternalization of the ferrisiderophore complex is crucial to the survival of the bacterial

cell.5, 6

In order to study siderophore-based iron acquisition systems of B. anthracis in the

most efficient manner possible, the availability of research quantities of petrobactin

becomes a legitimate concern. Typically, siderophores are isolated from supernatants of

bacterial cultures and purified several times to obtain a product worth of research.7

Because of the complexity of this process, overall yield can be low, so researchers must

2

extract from large cultures of bacteria to obtain appreciable amounts of the material.

When dealing with highly pathogenic B. anthracis, siderophore isolation methodology

can pose significant health risks to researchers.8

In order to address these problems, members of the scientific community have

since devised methods to produce petrobactin utilizing both enzymatic and chemical

synthesis techniques.9-13 These techniques, however, involve difficult, inefficient

reactions that require refining in order to improve overall petrobactin yield. Consequently,

we chose to undertake the task of optimizing petrobactin chemical synthesis so

quantifiable amounts of the siderophore can be produced in a reasonably straightforward,

convergent synthesis.14

3

1.3 Experimental Section

1.3.1 Chemicals and Materials

Spectrograde solvents were used without further purification. Anhydrous DCM,

DMSO, DMF, EtOAc, MeOH, H2SO4, methanesulfonyl chloride, 3-(dibenzylamino)-1-

propanol, N-Boc-1,4-butanediamine, 3,4-dihydroxybenzoic acid, PhSiH3, DIPEA, TEA,

triphenylphosphine, and (Ph3P)4Pdo were purchased from Sigma Aldrich (St. Louis, MO).

Antimony (III) ethoxide was purchased from ProChem, Inc. (Rockford, IL). PyBOP was

purchased from GenScript (Piscataway, NJ).

1.3.2 Equipment

All NMR spectra were recorded on either 400 MHz Varian or 500 MHz Bruker

spectrometers.

1.3.3 Synthesis of 3-(dibenzylamino)propyl methanesulfonate (3)

Methanesulfonyl chloride was added dropwise to the cooled solution of 3-

(dibenzylamino)-1-propanol (2) and TEA in DCM. The mixture was stirred at 0oC. After

completion of the reaction (2 h) water was added and the crude mixture was extracted

with DCM, washed with brine, dried over anhydrous Na2SO4 and concentrated. The

residue was purified by column chromatography (NH- silica gel, hexanes/EtOAc 9.5:0.5)

to afford 3 as a colorless gum.

4

1.3.4 Synthesis of tert-Butyl (4-((3-(dibenzylamino)propyl)amino)butyl)carbamate (5)

A mixture of 3, N-Boc-1,4-butanediamine (4) and DIPEA in dry DMSO was

stirred at 80oC under nitrogen environment for 24 h. The reaction was quenched with

water. The layers were separated and the aqueous layer was extracted several times with

EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, and

concentrated. The concentrated residue was purified by column chromatography (NH-

silica gel, hexanes/EtOAc 9.5:0.5 to 7:3) to afford 5 as a colorless gum.

1.3.5 Synthesis of tert-butyl (4-((3-aminopropyl)amino)butyl)carbamate (6)

A mixture of 5 and Pd/C (10%) in ethanol was refluxed under H2(g) for 4 h. The

suspension was filtered over celite and the filtrate was concentrated to give the crude

product, 6, which was used in the next step.

1.3.6 Synthesis of methyl 3,4-bis(benzyloxy)benzoate (9)

To a solution of 3,4-dihydroxybenzoic acid (7) in MeOH was added a catalytic

amount of concentrated H2SO4 and the mixture was refluxed for 8h. The reaction mixture

was cooled to room temperature and solvent was removed by rotary evaporation. The

remaining residue was dissolved in EtOAc and washed with saturated NaHCO3 and brine.

The combined extract were dried over Na2SO4, and concentrated to give the crude methyl

ester 8. To the residue was added a solution of benzyl bromide and K2CO3 in DMF. The

mixture was heated at 120oC. After 36 h, the reaction mixture was cooled to ambient

temperature and poured into ice-cold water. The mixture was then passed through cotton

5

to remove K2CO3. The mixture was extracted with EtOAc 3 times. The organic layers

were washed with ice-cold 1 M HCl, water, brine, dried over Na2SO4, and concentrated.

The residue was purified by column chromatography (silica gel, hexanes/EtOAc 9.5:0.5)

to afford 9 as a colorless gum.

1.3.7 Synthesis of tert-Butyl (4-((3-(3,4-bis(benzyloxy)benzamido)propyl)amino)butyl)carbamate (10)

To a mixture of 6, 9 was added with Sb(OEt)3 in neat conditions at room

temperature and the flask was purged with N2(g). The mixture was heated at 80oC for 24

h. After cooling to room temperature, the mixture was quenched with MeOH and filtered

through pad of celite using CHCl3, MeOH, then i-PrNH2. The resulting filtrate was

concentrated with a rotary evaporator and the resulting residue was purified by column

chromatography on Cromatorex®-NH-DM1020 silica gel using a mixture of

EtOAc:MeOH (100:0 to 100:1) as a mobile phase to give 10.

1.3.8 Synthesis of N-(3-((4-aminobutyl)amino)propyl)-3, 4-bis(benzyloxy)benzamide (11)

TFA was added dropwise to the cooled solution of 10 in DCM. The mixture was

stirred at 0oC. After completion of reaction (2 h), reaction mixture was concentrated and

it was used as the TFA salt without any purification.

6

1.3.9 Synthesis of 2-tert-Butyl-1,3-di-N-(hydroxyl)-Succinimidyl Citrate (13)

13 was synthesized in 4 steps from citric acid (12) according to previously

described procedures.15

1.3.10 Synthesis of tert-butyl 4-((4-((3-(3,4-bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-(2-((4-((3-(3,4-

bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-oxoethyl)-2-hydroxy-4-oxobutanoate (14)

11 was dissolved in dioxane and DCM and TEA was added to the stirring flask to

facilitate deprotection of the TFA salt. 13 was then added to the flask and the reaction

was allowed to proceed for 1.5 h after which the solvent was removed by rotary

evaporation. The residue was dissolved in EtOAc and extracted 3 times with H2O. The

organic layer was isolated and dried over MgSO4. The mixture was filtered through

cotton and the filtrate was concentrated using rotary evaporation to yield 14.

1.3.11 Synthesis of Petrobactin (1)

14 was dissolved in DCM/H2O (100:1) and the mixture was cooled to 0°C. TFA

was added to the solution and the reaction was allowed to warm to room temperature.

After 2 h, the solvent was removed by rotary evaporation, and placed under high vacuum

to ensure complete removal of TFA. Benzylated petrobactin was recovered and

redissolved in a mixture of AcOH/H2O (20:1) in a flask purged with H2. 10% Pd/C was

added to the flask and the reaction was stirred for 2h under H2. The mixture was filtered

through cotton and the filtrate was extracted 3 times with EtOAc. The pH of the aqueous

7

layer was neutralized with 1M K2CO3 and the resulting solution was lyophilized to yield

petrobactin (1).

1.4 Results and Discussion

To synthesize 1 (see figure 1.1), we designed a convergent synthesis using 3

building blocks that were individually modified and protected for later assembly. In order

to prepare the first building block, outlined in Scheme 1.1, we started with commercially

available 2, which was O-mesylated, providing the terminal hydroxyl with an adequate

leaving group for subsequent nucleophilic substitution. The mesylated product, 3, was

reacted with the mono-protected diamine, 4, to yield a protected version of the first

building block, compound 5. In order to deprotect the terminal amine group, a

debenzylation reaction was performed using catalytic hydrogenation to yield 6 as a free

primary amine.

Scheme 1.2 demonstrates preparation of the protected dihydroxybenzoic acid

moeity required for petrobactin synthesis. Commercially available 7 was refluxed in

H2SO4/MeOH to esterify the aryl carboxylic acid, yielding 8. The catechol component of

compound 8 was then protected using benzyl bromide to produce compound 9.

Assembly of these two building blocks was performed in the first step of Scheme

1.3, where 6 and 9 were coupled using Sb(OEt)3 by heating the reaction in neat

conditions . This important step in petrobactin synthesis notoriously produces low-yields

of 10 using standard peptide-coupling reagents, so we modified the reaction to include a

high-yielding Sb(OEt)3-mediated ester amide transformation reaction based on

procedures outlined in the literature.12, 16 Boc was removed from the terminal amine with

T

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9

10

CHAPTER 2. CHEMICAL SYNTHESIS OF STAPHYLOFERRIN A AND ITS EVALUATION FOR USE IN A PATHOGEN-DETECTION DEVICE

2.1 Abstract

Siderophores are unique molecules that can be used to capture bacteria that

express a specific receptor for them. In order to test this hypothesis, we synthesized a

siderophore unique to S. aureus called staphyloferrin A, and stamped it in a distinctive

pattern on a gold chip using microcontact printing. Upon introduction of the chip to a

solution containing S. aureus, capture of the pathogen was witnessed within the confines

of pattern where SA was stamped. These experiments provide evidence of an interaction

that occurrs between S aureus and SA. These findings are significant because they imply

that siderophores are capable of capturing bacteria on a small device that can be imaged

using optical microscopy.

2.2 Introduction

S. aureus is a gram-positive bacterium that commonly resides on human epithelial

tissues and nasal mucosa. It is a community microbe that thrives in environments with

unsanitary conditions, and is believed to be a primary causative factor of an estimated

500,000 surgical site infections of hospital patients in the United States alone.17 Among

the most pathogenic of these infections are caused by MRSA and VRSA – antibiotic-

11

resistant mutants of S. aureus that require rigorous and potentially toxic treatment

regimens.18-20

In order to effectively treat nosocomial pathogens like S. aureus, we must focus our

priorities on early detection. The ability to diagnose communicable diseases in the early

stages of progression is a powerful tool we have against prohibiting rapid transmission of

the disease throughout exposed populations. Diagnosis involves isolation of the bacteria,

pre-enrichment, and characterization using RT-PCR, a process that consumes a

significant amount of time that could be spent treating the patient and preventing disease

communication.21

To expedite the process of pathogen detection and identification, we have

developed a device with the sole purpose of specifically capturing S. aureus. It is a lab-

on-a-chip device that can be imaged with a benchtop optical microscope at low

magnification.22, 23 The device was designed to allow for clear distinction between a

blank chip and a chip containing captured bacteria. With dark field microscopy, the

bacteria can be easily detected as they scatter light and appear as bright yellow spots

against a black background.

It is exceedingly important that this device is able to capture S. aureus with a high

degree of specificity, since the capturing of other strains of bacteria can give misleading

data to clinicians, which can effectively lead to disease misdiagnosis and subsequent

mistreatment. We attempted to overcome this problem by taking advantage of a particular

interaction that occurs between the ABC transporter HtsA, located on the outer

membrane of S. aureus, and its cognate siderophore SA.24-26

12

Siderophores are small organic chelators that bacteria biosynthesize and release

into their extracellular environment for sequestration of ferric iron.27 The iron-

siderophore chelate is reinternalized by bacterial cells using a specialized receptor

complex. Once inside the cell, Fe(III) is reduced to Fe(II) and released from the

siderophore to be incorporated into essential metabolic processes.28 Herein, we wish to

report on the methods developed to chemically synthesize SA using SPPS, and the

implementation of the siderophore in a pathogen detection device.22

2.3 Experimental Section

2.3.1 Chemicals and Materials

Fmoc-D-Ornithine(Alloc)-OH was purchased from Iris Biotech (Marktredwitz,

Germany). Dichloromethane (DCM), methanol (MeOH), and dimethylformamide (DMF)

were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Triphenylphosphine (PPh3),

tetrahydrofuran (THF), diisopropyl azodicarboxylate, Stratospheres™ PL-Wang resin,

piperidine, diisopropylethylamine (DIPEA), phenylsilane (PhSiH3),

tetrakis(triphenylphosphine)palladium ((Ph3P)4Pd0), citric acid, trifluoroacetic acid (TFA),

iron(III) chloride, bovine serum albumin (BSA), phosphate-buffered saline (PBS), and

2,2’-bipyridine were purchased from Sigma-Aldrich (Saint Louis, MO). Benzotriazol-1-

yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from

GenScript (Piscataway, NJ). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was

purchased from Bio-Rad (Hercules, CA).

13

2.3.2 Equipment

All solid-phase synthesis reactions were performed in a solid-phase peptide

synthesis vessel from Chemglass Life Sciences (Vineland, NJ). Hilic Atlantis Silica

HILIC OBD Preparative HPLC column was purchased from Waters Corporation

(Milford, MA). Gold surface plasmon resonance (SPR) chips were purchased from

Reichert Technologies (Depew, NY). All images were taken with an Olympus BX43

LED Upright Microscope.

2.3.3 Synthesis of Staphyloferrin A22

Fmoc-D-Orn(Alloc)-OH and triphenylphosphine were added to DCM:THF (1:1),

and the solution was cooled to 0°C in an ice bath. DIAD was added dropwise to the

solution to prevent generation of excess heat. The solution was then added to a SPPS

vessel containing StratoSpheresTM PL-Wang resin, 100–200 mesh that was pre-swollen

in DCM for 10 min prior to addition. Ar(g) was bubbled through the solution for 36 h,

allowing maximum loading of the residue onto the resin. The solution was removed from

the SPPS vessel and the resin was washed with 3 x 5 mL DCM, 3 x 5 mL THF, 3 x 5 mL

DMF, and 3 x 5 mL DCM. The resin was treated with a solution of 20% piperidine in

DMF to remove the N-terminal Fmoc (3 x 5 mL) over the course of 30 minutes. The

Fmoc-cleavage solution was drained from the vessel and the resin was washed with 3 x 5

mL DMF. After verifying the presence of a primary amine on the residue with a Kaiser

test, a solution of 3-t-Bu-citrate,15 PyBOP, and DIPEA in 4 mL DMF was introduced to

the vessel. The reaction was bubbled with Ar(g) for 12 h. After removing the coupling

solution and washing the resin with 3 x 5 mL DMF, 3 x 5 mL MeOH, and 3 x 5 mL

14

DCM , a Kaiser test was used to determine completion of the reaction by signifying the

lack of a free primary amine on the resin. Next, a solution of (Ph3P)4Pd0 and PhSiH3 in

DCM was added to the resin to remove Alloc from the ornithine amine. The reaction was

allowed to bubble with Ar(g) for 2 h, the solution was drained, and the resin was washed

with 3 x 5 mL DCM, 3 x 5 mL DCM:DIPEA (19:1), 3 x 5 mL DMF, and 3 x 5 mL DCM.

A positive Kaiser test affirmed the presence of a primary amine on the resin. A solution

of 3-t-Bu-citrate, PyBOP, and DIPEA in DMF was introduced to the vessel. The reaction

was bubbled with Ar(g) for 12 h to ensure reaction completion. After removing the

coupling solution from the vessel, the resin was washed with 3 x 5 mL DMF, 3 x 5 mL

MeOH, and 3 x 5 mL DCM. A negative Kaiser test indicated that the reaction had

reached completion. In order to cleave SA from the resin and simultaneously deprotect

tert-butyl groups present on citrate moieties, a solution of 92.5% TFA, 2.5% DCM, 2.5%

H2O and 2.5% TIPS was added to the vessel and allowed to bubble with Ar(g) for 1 h.

The cleavage solution was drained into a round bottom flask, and combined with two

additional portions of the cleavage solution used to wash the resin remaining in the vessel.

The cleavage solution was concentrated under reduced pressure to approximately 2 mL,

making certain to avoid heating the solution beyond 40° C while evaporating.10 To the

concentrate was added diethyl ether and the precipitate was collected by centrifugation.

After decanting the supernatant, the precipitate was washed with diethyl ether and

centrifuged three additional times to remove any remaining ether-soluble components.

The precipitate was dried under high vacuum to yield the crude mixture of diastereomers

as white powder. The material was purified by preparative HPLC using an Atlantis Silica

HILIC OBD column (19 x 150 mm, 5 μm) with a 10 mM ammonium acetate buffer (pH

15

7) and acetonitrile mobile phase yielding two chromatographically distinguishable

diastereomers of SA.

2.3.4 Preparation of Staphyloferrin A-BSA Conjugate

14 mmol of SA was dissolved in deionized water and gently stirred as a 0.2 M

aqueous FeCl3 solution was added dropwise. The chelation reaction was allowed to stir

for 5 min and lyophilized. In order to prepare “ink” used for PDMS stamping, 96 μmol of

EDC was combined with 28 μmol of staphyloferrin A-Fe in 375 μL of pH 7.4 PBS. This

mixture was stirred at room temperature for 30 minutes to activate the ornithine

carboxylate. A 1 mL solution of 0.9 mmol (60 mg) BSA in PBS was prepared and added

to the reaction and stirred for an additional 2 h. After completion of the reaction, the

mixture was filtered using 0.45 μm membrane filter. The filtrate was collected and either

used for immediate PDMS stamping, or lyophilized for later use.

2.3.5 Preparation of Staphyloferrin A-BSA-patterned Chips

To produce a PDMS stamp that will create a pattern visible by low magnification

optical microscopy, a silicon wafer was etched with 20 μm wide parallel lines with 20 μm

gaps using UV photolithography. The stamp was fabricated by pouring viscoelastic liquid

PDMS over the silicon master and allowing it to cure. The hardened PDMS was

henceforth able to be used for microcontact printing applications. To apply ink to the

stamp, a cotton swab was dipped in 50 uL of SA-BSA ink, rolled onto the textured side

of the PDMS, and allowed to dry in the inverted position after removing excess liquid

with a dry cotton swab. The inked stamp was then placed on a gold-coated glass chip,

16

pressed lightly for 10 seconds, and allowed to sit on top of the chip for an additional 5

minutes. The stamp was carefully removed and the chip was sealed in a sterile Petri dish

for 5 hours at 4°C. To block portions of the chip unstamped by the PDMS, a 5% (w/w)

BSA solution was prepared in pH 7.4 in PBS and applied to the chip for 5 minutes. The

BSA solution was removed with a sterile pipette and the chip was rinsed thoroughly with

3 x 5 mL portions of pH 7.4 PBS.

2.3.6 Growth of S. aureus

A frozen stock of S. aureus was used to seed a growth culture in standard LB

broth at 37°C on an orbital shaker overnight. 10 μL of the seed culture was transferred to

low-iron LB media, which was prepared by adding 2,2′-bipyridine to a concentration of

0.2 mM. The bacteria were grown to a concentration of 1 x 108 CFU/mL by measuring

optical density at 600 nm with a spectrophotometer and diluted with pH 7.4 PBS to the

appropriate concentration for experiments.

2.3.7 Capture and Imaging of Chips

50 uL of 1 x 106 CFU/mL S. aureus was dispensed onto siderophore-stamped

gold chips and incubated in a closed, sterile Petri dish for 1 h. The solution was removed

with a sterile pipette and the chip was rinsed thoroughly with 3 x 5 mL pH 7.4 PBS and

deionized water respectively. The chips were dried with a gentle stream of Ar(g) to

remove any excess water. Dry chips were imaged using dark field optical microscopy at

10x magnification.

17

2.4 Results and Discussion

In order to exploit the receptor-ligand interaction that exists between SA and

HtsA, we chose to design a synthesis scheme which allowed us to chemically produce SA.

Prior to this study, the siderophore was obtained through isolation from live S. aureus, a

laborious, time-consuming process requiring growth of a large amount of bacteria,

extraction from the bacterial supernatant, and multiple purifications.29 By synthesizing

SA, we are able to considerably simplify this process by using dependable and high-

yielding peptide-coupling strategies on solid-phase resin. Synthesizing the ligand on solid

phase eliminates the need for purification after each reaction, a compulsory practice

associated with conventional solution-phase syntheses. This approach also allows us to

circumvent purification concerns associated with contamination from other chemically-

similar siderophores or small molecules produced by S. aureus that are normally present

in a crude culture supernatant.

Scheme 2.1 demonstrates how SA can be methodically synthesized using SPPS

techniques. After cleavage from the resin and HPLC purification, SA was chelated to

Fe3+ and simultaneously reacted with BSA using traditional EDC-coupling techniques in

an aqueous medium.

A dilute solution of SA-BSA conjugate was stamped onto gold chips using a

patterned PDMS stamp. Portions of the chip where the pattern was not present were

blocked with BSA to ensure there was no reactive gold exposed on the chip. The chip

was exposed to a solution of 106 CFU/mL S. aureus in order to examine its ability to

capture the pathogen for the purpose of identification. The chip was later imaged using

dark field microscopy at 10x magnification. Capture was witnessed and is exemplified in

18

figure 2.1. When compared to images taken of the same chip prior to exposure to S.

aureus, captured bacteria are readily observable as bright yellow clusters that contrast

against the dark background of the chip. Since the SA-BSA conjugate was stamped in a

specific pattern and the capture reflects this pattern, it is discernible that there is no

observable interaction between S. aureus and BSA alone, but exclusively between the

siderophore and its cognate receptor on the cell surface of the pathogen.

2.5 Conclusion

Though the siderophore-patterned chip shows promise as an effective means of

pathogen capture and detection in these and other preliminary studies,23, 30 additional

experiments are required to investigate the limit of detection, the specificity towards S.

aureus versus other human pathogens, and the minimum amount of time required for

discernable capture.

We have learned that the chemical synthesis of SA and the production of the

pathogen capture device are relatively inexpensive and straightforward processes. It can

also be suggested that this method of pathogen detection may significantly reduce the

amount of time required to assay for a specific strain of bacteria when compared to

commonly accepted methods in the literature.31, 32 This is an exceedingly important

characteristic for a pathogen detection system, since rapidly-communicable diseases can

disseminate through a population in a very short period of time. In addition to these

benefits, now that a simple, high-yielding chemical synthesis of SA has been developed,

its other potential applications can be investigated without the need for time-consuming

culture isolation methodology.26, 33, 34 Because of this, we anticipate the use of SA in a

19

variety of other applications, ranging from its use as a constituent in targeted antibiotic

delivery constructs, to its potential function as a metal chelator for use in patients with

iron-overload disorders.35-41

Scheme 2.1. Chemical synthesis of SA using SPPS techniques.22

HO

WangResin-OH

AllocHN O

O

NH

Fmoc-D-Orn(alloc)-OH

DIAD, PPh330 h

20% piperidine in DMF

30 min

AllocHN O

O

NHFmoc

92.5% TFA, 2.5% H2O,

2.5% DCM, 2.5% TIPS1 h

HN O

O

HNO

tBuOOC

OOH

OHO

OHO

tBuOOC

(PPh3)4Pdo, PhSiH3,

DCM

2 h

HN OH

O

HNO

HOOC

OOH

OHO

OHO

HOOC

O

tBuOOC

OOH

OH

OH OH

AllocHN O

O

NH2

PyBOP, DIPEA, DMF12 h

H2N O

O

NH

O

tBuOOC

OOH

OH

HOO

COOtBu

OHO

HO

PyBOP, DIPEA, DMF12 h

HOO

COOtBu

OHO

HO

Figure 2.1stamped omagnifica

. Capture of on a gold subation. (Left)

f S. aureus usbstrate. Imag

Prior to S. a

sing a lab-onges were takeaureus captu

n-a-chip deven using dar

ure. (Right) A

vice consistinrk field micrAfter S. aure

ng of SA-BSoscopy at 10eus capture.2

20

SA 0x 22

21

CHAPTER 3. SELECTIVE CAPTURE OF S. AUREUS USING IMMOBILIZED SIDEROPHORE STAPHYLOFERRIN A

3.1 Abstract

The necessity for a quick and inexpensive diagnostic tool for bacterial disease has

become increasingly essential as dangerous mutants arise through antibiotic resistance

mechanisms and total eradication of some pathogenic strains remains difficult.17-19, 42, 43

In order to address this problem, we developed a pathogen detection device that is

capable of rapidly and specifically capturing S. aureus at concentrations as low as 1 x 102

CFU/mL. This technology takes advantage of an innate high-affinity interaction that is

present between HtsA, a membrane-bound transporter on S. aureus, and staphyloferrin A,

a unique S. aureus siderophore called that has been immobilized on a gold chip.22, 26, 33

3.2 Introduction

Population mobility is an increasingly important concern when it comes to the

rapid spread of communicable diseases throughout the world. Advances in transportation

technology have allowed us to quickly and easily translocate, dramatically increasing the

probability of major disease outbreaks and pandemics.44 Consequently, researchers and

clinicians must lead the movement to develop pathogen detection devices that are not

only capable of specific pathogen identification, but are also inexpensive, simple to use,

and easy to obtain anywhere in the world.

22

To achieve this, we have constructed a lab-on-a-chip device consisting of an

immobilized, S. aureus-specific ligand that captures the pathogen via a receptor-ligand

interaction. The ligand, a siderophore called SA, is biosynthesized by S. aureus, and is

released into the extracellular environment to sequester ferric iron, The ferrisiderophore

complex is then reinternalized by the bacteria, where captured iron can be incorporated

into essential intracellular redox processes.24-27, 34

Because there is a unique interaction between SA and HtsA, a membrane-

spanning ABC transporter responsible for uptake of the ferrisiderophore complex, we

designed a lab-on-a-chip device that uses support-bound SA to capture S. aureus. The

siderophore was loaded onto BSA, which was then reacted with the gold surface of the

chip in a predetermined pattern specified by microcontact PDMS stamping. S. aureus can

be captured by SA present on the chip, and the bacterial cells can be imaged using dark

field microscopy.

After the first solid-phase chemical synthesis of SA was published by our

laboratory in 2014,22 we chose to expand upon this procedure by adding a peptidic spacer

(15) that should improve siderophore loading onto BSA and S. aureus capture efficiency.

We also undertook the task of evaluating the siderophore’s ability to selectively capture

the pathogen in a variety of conditions and exposure times.

23

3.3 Experimental Section

3.3.1 Chemicals and Materials

Fmoc-D-Ornithine(Alloc)-OH was purchased from Iris Biotech (Marktredwitz,

Germany). Dichloromethane (DCM), methanol (MeOH), and dimethylformamide (DMF)

were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Triphenylphosphine (PPh3),

tetrahydrofuran (THF), diisopropyl azodicarboxylate, Stratospheres™ PL-Wang resin,

piperidine, diisopropylethylamine (DIPEA), phenylsilane (PhSiH3),

tetrakis(triphenylphosphine)palladium ((Ph3P)4Pd0), citric acid, trifluoroacetic acid (TFA),

iron(III) chloride, bovine serum albumin (BSA), phosphate-buffered saline (PBS), and

2,2’-bipyridine were purchased from Sigma-Aldrich (Saint Louis, MO). Benzotriazol-1-

yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from

GenScript (Piscataway, NJ). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was

purchased from Bio-Rad (Hercules, CA).

3.3.2 Equipment

Solid-phase peptide synthesis vessels from Chemglass Life Sciences (Vineland,

NJ) were used in all solid-phase synthesis reactions. Atlantis Silica HILIC OBD

Preparative HPLC column was purchased from Waters Corporation (Milford, MA). Gold

surface plasmon resonance (SPR) chips were purchased from Reichert Technologies

(Depew, NY). Images were taken with an Olympus BX43 LED Upright Microscope.

24

3.3.3 Synthesis of 1522

To a SPPS vessel containing 2-chlorotrityl resin pre-loaded with H-Cys(Trt),

DCM was added and bubbled with N2(g) to swell the resin. The solvent was drained, and

a solution of 20% piperdine in DMF was added to the vessel and bubbled for 30 min to

remove the terminal Fmoc. The solution was drained and the resin was washed with 3 x 5

mL DMF, 3 x 5 mL MeOH, and 3 x 5 mL DCM. A solution of Fmoc-L-Asp(OtBu)-OH,

PyBOP, and DIPEA in DMF was added to the vessel and bubbled with N2(g) for 5 h. The

reaction mixture was drained and the resin was washed with 3 x 5 mL DMF, 3 x 5 mL

MeOH, and 3 x 5 mL DCM. This process of Fmoc deprotection, peptide coupling, and

extensive washing was repeated with the following residues: Boc-L-Dap(Fmoc)-OH,

Fmoc-Glu(OtBu)-OH, Fmoc-D-Orn(Alloc)-OH and the first 3-tBu-citrate moiety. A

solution of (PPh3)4Pdo and PhSiH3 in DCM was then added to the reaction vessel to

deprotect the Alloc-protected ornithine amine. The resin was washed and reacted with the

second 3-tBu-citrate moiety using the same conditions as prior peptide couplings. To

cleave compound 1 from the resin, a solution of 95 % TFA, 2.5% ethanedithiol, and 2.5%

TIPS was added to the resin and bubbled with N2(g). The cleavage mixture was collected

and concentrated by rotary evaporation at room temperature.10 A white powder was

precipitated from the crude concentrate using diethyl ether and the precipitate was

washed with additional portions of diethyl ether. The white powder was purified by

HPLC using a Waters Atlantis Silica HILIC OBD Preparative HPLC column with 10

mM ammonium acetate buffer (A) and acetonitrile (B) (95%B - 65%B) over 30 min.

25

3.3.4 Synthesis of 15-BSA conjugate

Refer to section 2.3.4 for details.

3.3.5 Preparation of 15-BSA-patterned chips

See figure 3.2 and section 2.3.5 for details.

3.3.6 Growth of S. aureus

Refer to section 2.3.6 for details.

26

3.3.7 Capture and Imaging of Chips

3.3.7.1 Evaluation of Limit of Detection

50 μL of S. aureus in pH 7.4 PBS was dispensed onto siderophore-patterned gold

chips in a sterile Petri dish at 1 x 106, 1 x 105, 1 x 104, 1 x 103, or 1 x 102 CFU/mL and

incubated for 1 h. The chips were then washed with 3 x 5 mL pH 7.4 PBS and 3 x 5 mL

deionized water to remove any unbound bacteria from the chip. The chips were then

imaged using dark field microscopy at 40x magnification.

3.3.7.2 Time-Course Study

50 μL of 1 x 106 CFU/mL S. aureus in pH 7.4 PBS was dispensed onto

siderophore-patterned gold chips in a sterile Petri dish and incubated for the following

times: 5 min, 30 min, 1 h, 2 h. Afterwards, the chips were washed with 3 x 5 mL pH 7.4

PBS and 3 x 5 mL deionized water to remove any unbound bacteria from the chip. The

chips were imaged using dark field microscopy at 40x magnification.

27

3.3.7.3 Evaluation of Chip Specificity

50 μL of pH 7.4 PBS containing 1 x 106 CFU/mL S. aureus, E. coli, P. aeruginosa, 

S. flexneri, V. cholerae, Y. enterocolitica, or S. enterica was dispensed onto siderophore-

patterned gold chips in a sterile Petri dish and incubated for 1 h. Afterwards, the chips

were each washed with 3 x 5 mL pH 7.4 PBS and 3 x 5 mL deionized water to remove

any unbound bacteria from the chip. The chips were imaged using dark field microscopy

at 10x magnification.

3.4 Results and Discussion

Compound 15 was produced using established solid-phase synthesis techniques,

and the synthetic scheme we developed was based on a similar synthesis of SA published

by our laboratory in 2014.22 Our recent findings have provided us with insight on how to

produce high yields of enantiomerically pure 15 (see figure 3.1).

Beginning with preloaded H-Cys(Trt)-2-chlorotrityl resin, we systematically

added Fmoc-L-Asp(OtBu)-OH, Boc-L-Dap(Fmoc)-OH, and two Fmoc-Glu(OtBu)-OH

residues to the resin respectively using SPPS strategies. SA was modularly synthesized at

the N-terminus of the peptidic spacer with addition of differentially deprotectable Fmoc-

Orn(Alloc)-OH and two 3-t-Bu-citrate residues. Compound 15 was cleaved from the

resin, purified by HPLC, and used in pathogen detection studies in order to assess its

ability to capture S. aureus.

To expand upon previously-published pathogen capture studies conducted using a

lab-on-a-chip device,23, 30, 45 15 was covalently coupled to BSA, mediated by EDC in pH

28

7.4 PBS. This solution was used as ink for microcontact printing in order to produce a

specific pattern of 15-BSA on a gold chip according to figure 3.2 below. The pattern,

imprinted on a PDMS stamp from a silicon master, consists of raised 20 μm wide lines in

parallel with 20 μm gaps, producing a recognizable pattern that is observable with low-

magnification optical microscopy.

After stamping, the gold chips were blocked with a solution of unlabeled BSA,

which reacts with gold exposed on the non-stamped components of the chip. This helps

avoid any potential non-specific interactions between bacteria and the oxidized gold

surface of the chip.45-47 The originally-stamped pattern of 15-BSA was indistinguishable

from BSA used for blocking, but was observable in the following experiments by

imaging S. aureus captured in the same pattern.

To determine the limit of detection (LoD) of this device, we exposed stamped

chips to incrementally decreasing concentrations of S. aureus ranging from 1 x 106

CFU/mL to 1 x 102 CFU/mL. Using dark field microscopy, we imaged the chips at 40x

magnification. Figure 3.3 exhibits visible capture of S. aureus down to 1 x 102 CFU/mL.

This data suggests that future applications of this device may enable early detection of S.

aureus-related infections since pathogens can be effectively detected at early stages of

population propagation without the necessity for pre-enrichment cultures.21 In addition to

having a low limit of detection, the device also proved to capture the pathogen in an hour

or less of exposure time, which is a vast improvement over other pathogen-detection

methods like RT-PCR.48-50 Results of the time-course experiment are shown in figure 3.4.

Upon examination of the specificity of the chip when screened against a panel of

other human pathogens at 1 x 106 CFU/mL, there was clear evidence of S. aureus capture

29

(see figure 3.5). Aside from a low degree of cross reactivity exhibited with S. enterica,51

our construct did not capture any of the other bacterial strains tested. The high degree of

specificity that exists between compound 15 and S. aureus is an important characteristic

that contributes to the simplicity and reproducibility of the assay by discriminating

against false-positive results.

3.5 Conclusion

When taken together, the data collected from these experiments indicate that this

device, which can selectively detect low concentrations of a S. aureus in minimal time,

has enormous potential in the field of medicine. We expect that this device will accelerate

the process of detecting S. aureus in our environment, and hope that it will contribute to

the process of eradicating this dangerous pathogen from affected areas.

Figure 3.1. CChemical strructure of feuse in

erric-staphylon capture stu

oferrin A wiudies (15).

ith a short peeptide linker

30

r for

31

Scheme 3.1. Solid-phase synthesis of 15.

F

Figure 3.2. TThe microcontact printinng process usa-chip devi

sed on gold cice.

chips to prod

duce our lab

32

b-on-

FFigure 3.3. C(B) 1 x 105 C

(F) BS

Figure 3the chi

Capture efficCFU/mL, (C

SA only with

3.4. Time-cop. (A) 2 h, (B

iency based C) 1 x 104 CFh 1 x 106 CFU

ourse study toB) 1 h, (C) 3

on concentrFU/mL, (D) U/mL S. aurmagnificati

o examine th30 min, (D)

magnificat

ration of S. a1 x 103 CFU

reus. All imaion.

he effect of b5 min. All imtion.

aureus. (A) 1U/mL, (E) 1 ages were tak

bacterial expmages were

1 x 106 CFUx 102 CFU/mken at 40x

posure time ttaken at 40x

33

U/mL, mL,

to x

FFigure 3.5. S(A) Before

choler

pecificity ofe capture, (Brae, (G) Y. e

f capture dev) S. aureus,

enterocolitica

vice when te(C) E. coli, a, (H) S. ent

magnificat

ested against (D) P. aerug

terica. All imtion.

a variety ofginosa, (E) Smages were t

f human pathS. flexneri, (Ftaken at 10x

34

hogens. F) V.

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35

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