Detecting ‘Heat’ with Light: Luminescent Sensors for Capsaicin

Christopher G. Gulgas

N N

O

NHO

OOO

OHN

Ln3+

H3CO

HO

NH

O

Chemistry review: Hydrogen bonding

• Molecules with O-H bonds or N-H bonds can “hydrogen bond” to other molecules

HO

H δ+

δ-

δ+

HO

H

Chemistry review: Hydrogen bonding

O

N

H

N

O

N

HH

H

O Hδ- O

N

H

δ-

• Lipids are hydrophobic molecules usually containing long carbon chains mostly saturated with hydrogen

• They are nonpolar molecules and would disrupt the hydrogen-bonding network in bulk water

Chemistry review: LipidsO

HO

Palmitic acid

O

O

O

O

O

O

Triglyceride based on palmitic acid

Part 1 - Capsaicin

H3CO

HO

NH

O

= area of (+) charge= area of (-) charge

“oily” alkyl chain (not rigid)

Capsaicin

• Member of the vanilloids

• Biological properties1-2

– Anticancer– Antioxidants– Analgesic (pain relief creams)– Anti-inflammatory– Antimicrobial– Promote energy consumption and

decrease accumulation of fats

H3CO

HO

NH

O

H3CO

HO

O

O

H3CO

HO

O

HH3CO

HO

O

capsaicin (hot peppers)

capsiate (sweet peppers)

vanillin (vanilla) zingerone (ginger root)

1. Barbero, G.F. et al. J. Agric. Food Chem. 2010, 58, 3342-3349.2. Rusterholz, D.B. J. Chem. Educ. 2006, 83, 1809-1815.

Naturally-occurring capsaicinoids• Capsaicin

• Dihydrocapsaicin

• Nordihydrocapsaicin

• Homocapsaicin

• Homodihydrocapsaicin

H3CO

HO

NH

O

H3CO

HO

NH

O

H3CO

HO

NH

O

H3CO

HO

NH

O

H3CO

HO

NH

O

What’s the effect of shortening the “oily” chain?

Capsaicin3

• Nelson evaluated pungency– Relative pungency determined by the noting the

minimum amount of capsaicinoid resulting in “a distinct burning on the tip of the tongue”.

– 0.00013 mg (130 ng) of capsaicin could be detected, and provided the standard for comparison

– Alcoholic solutions were made of the capsaicinoids, and concentrations were varied until the same degree of burning sensation was detected from one drop.

• Individual descriptions – n=0 (not pungent)– n=1 (very slightly pungent)– n=2 (somewhat pungent)– n=5 (far more pungent than earlier members)– n=6 (very pungent)– n=7 (violently pungent)

“about equal to capsaicin, but its property of causing sneezing and coughing is probably not quite so great”

– n=8 (extremely pungent – more disagreeable to handle than n=7)

– n=9 (extremely pungent; coughing and sneezing)– n=10 (different pungency, not so immediately

apparent, effecting the back of the tongue and throat)

H3CO

HO

NH

O

capsaicin (hot peppers)

3. Nelson, E.K. et al. J. Am. Chem. Soc. 1919, 41, 2121-2130.

H3CO

HO

NH

O

(CH2)nCH3

n = 0-11

capsaicin analogs (synthetic)

Comparative Pungencies

n=5 5

n=6 25

n=7 75

n=8 100

n=9 50

n=10 25

n=11 25

Hydrophobic effect

“fat/oily” region

HO

H=

Capsaicinoids as Pain Relievers2,4

• Capsaicin binds to the TRPV1 receptor protein (transient receptor potential vanilloid), an ion channel protein4

• TRPV1 is located in sensory nerve fibers throughout the body, activated by vanilloids or heat (> 42°C)

• Activation leads to an opening of the ion channel, and calcium appears to be the primary ion involved

• Acidic conditions enhance the effect• Desensitization to the effects of capsaicin

have been observed (with or without permanent damage to the nerve cells)

• Structural analogs to capsaicin have been synthesized, and their relative potency in binding to the TRPV1 receptor studied

• The goal is to design highly potent, less pungent desensitizing drugs to reduce pain

4. Caterina, M.J. et al. Nature 1997, 389, 816-824.

H3CO

HO

NH

O

RP = 0.55-0.71

H3CO

HO

HN

RP = 1.0O

H3CO

H3CO

NH

O

RP = 0.46

O

O

NH

O

RP = <0.01

H3CO

HO

N

O

RP = <0.01CH3

• Replacement of the amide functionality with a stronger hydrogen-bond donating group has resulted in higher relative potency (RP)

Capsaicinoids as Pain Relievers2

H3CO

HO

HN

RP = 0.016

H3CO

HO

NH

S

NH

RP = 5.0

O

H3CO

HO

NH

S

NH

O

RP ~ 500

O

Capsaicinoid synthesisH3CO

HO

O

H

vanillin

NH2OH HCl

NaOAc, MeOH

H3CO

HO

N

H

OH

H2 10% Pd/C

H3CO

HO

NH2

HCl

O

Cl2

3 N(et)3 ,CH2Cl2

H3CO

O

NH

O

Ovanillylamine HCl

KOH, MeOH

H3CO

HO

NH

O

cap1

83.9 %

98.7%68.0%

98.0% (55% Total)

Current Research Goal• Capsaicinoids are effective for pain relief, and have favorable biological

properties• Capsaicinoid concentrations can be measured readily by HPLC methods

• Questions• Can we design a luminescent sensor molecule for the detection of

capsaicinoids in solution?• Will undergraduate researchers learn structure-activity

relationships relating to drug discovery and design by synthesizing and evaluating these sensor molecules?

• Goal• I want to have a molecule (a metal complex) that detects the

capsaicinoid family through luminescence. Nothing exists like this for capsaicinoids.

• The creation of a suitable metal complex will open up further research in biological systems, potentially

• High Pressure Liquid Chromatography– Mixtures are separated and components are quantified– Separation occurs through molecular properties such as

polarity or size– Molecules interact with the column and the solvent– Can be expensive and time consuming, but is the industry

standard

Quantification of Capsaicin - HPLC

Example of data

2 3 4 5 6 7 8 90

0.5

1

1.5

2

x 105

Retention Time (min)

Abs

orba

nce

at 2

70 n

m (m

AU

)

Doxylamine

Guaifenesin Acetaminophen

Aspirin

Dextromethorphan

Caffeine

**Courtesy of Dr. Sarah Porter

A

Part 2 – Lanthanide-based Sensor

• A = analyte

N N

O

NHO

OOO

OHN

Ln3+

open coordination sites

EDTA

• Ethylenediaminetetraacetic acid• Usually incorporated in foods as “disodium EDTA”• Sequesters metal ions in paints/dyes, in foods, and in the body

N N

O

OHO

OOO

OHO

N N

O

OO

OOO

OO

N N

O

OHO

HOOHO

OHO

Na Na Na Na

Na Na

EDTA Disodium EDTA Tetrasodium EDTA

Lanthanide properties• Lanthanide (III) ions are hard acids,

preferring oxygen donors, and stable complexes are formed in aqueous solution only with multidentate ligands.

• Lanthanide ions typically coordinate 8-9 donor atoms

• Many lanthanides are luminescent in the visible range and have sharp emission lines

http://perso.univ-rennes1.fr/martinus.werts/lanthanides/ln_shine.html

HON

NN

OH

O O

O

OH

O

OH O

OH

DTPA

Lanthanide luminescence

– Direct excitation is not efficient; a light-harvesting antenna is typically used for augmented luminescence

– Millisecond lifetimes allow for time resolved fluorescence measurements

– Ln3+-antenna distance is important

– De-excitation of the excited lanthanide can occur through non-emissive pathway (OH, NH oscillators)

Antenna Spacer Ln3+

hv

hv2Energy Transfer

X-

H2O

Ln3+

H2O

H2O

Ln3+

X

XPetoud, S.; Cohen, S. M.; Bünzli, J.-C. G.; Raymond, K. N. J. Am. Chem. Soc. 2003, 125, 13324-13325.

Chelate Synthesis

NN

O

HO

HO O

OHO

OH

O

N

O

O

O

100 C

NN

O

O

O

O

OO

EDTA EDTA-BA

Ln(OTf)3 , 2 NaOH

Ln = Eu or Tb

OTf =O

S

O

CF3O

NN

O

HO

NH

O

OHO

HN

O

1

N N

O

NHO

OOO

OHN

Ln3+

[Ln(1)]+

40% CH3NH2

H2O

in H2ODMF

92%

42%

OO

HN

O

Binding capsaicinoids

N N

O

NHO

OOO

OHN

Eu3+

N N

O

NHO

OOO

OHN

Tb3+

OO

HN

O

OO

HN

O

OO

HN

O

How do we begin to understand what is going on?

Luminescence experiments will shed some light on the subject.

Luminescence Experiments• Emission Scan

– One wavelength of excitation

– Scan a range of wavelengths for emission intensity of the solution

• Excitation Scan– One wavelength of

emission measured– Use a range of

wavelengths for excitation of the solution

detector

sample

excitation source

detector

sample

excitation source

Proof-of-concept with cap1

• Over 500x luminescence augmentation!!

O

O

NH

O

cap1

N N

O

NHO

OOO

OHN

[Tb(1)]+

Tb3+

475 495 515 535 555 575 595 615 6350

100200300400500600700800900

1000

Titration of Tb-1 (290 exc) w/ cap-1

0 eq

1 eq

2 eq

3 eq

4 eq

5 eq

10 eq

Emission wavelength (nm)

Inte

nsity

(a.u

.)

475 495 515 535 555 575 595 615 63505

101520253035404550

Titration of Tb-1 (290 exc) w/ cap-1

0 eq

0.2 eq

0.4 eq

0.6 eq

0.8 eq

1 eq

Emission wavelength (nm)

Inte

nsity

(a.u

.)

[Tb(1)]+ = 5.0 x 10-5 M

0 eq 0.2 eq 0.4 eq 0.6 eq 0.8 eq 1 eq 2 eq 3 eq 4 eq 5 eq 10 eq 20 eq 30 eq 40 eq 50 eq

0

100

200

300

400

500

600

700

800

900

1000

Titration of Tb-1 (290 exc) w/ cap-1

621 nm585 nm489 nm545 nm

Inte

nsity

(a.u

.)

Excitation spectrum (545 emission)

O

O

NH

O

cap1

N N

O

NHO

OOO

OHN

[Tb(1)]+

Tb3+

250 270 290 310 330 350 370 3900

100200300400500600700800900

1000

Titration of Tb-1 (545 em) w/ Cap-1

0 eq1 eq2 eq3 eq4 eq5 eq10 eq

Excitation wavelength (nm)

Inte

nsity

(a.u

.)

250 270 290 310 330 350 370 3900

100200300400500600700800900

1000

Titration of Tb-1 (545 em) w/ Cap-1

10 eq20 eq30 eq40 eq50 eq

Excitation wavelength (nm)

Inte

nsity

(a.u

.)

[Tb(1)]+ = 5.0 x 10-5 M

0 eq 1 eq 2 eq 3 eq 4 eq 5 eq 10 eq 20 eq 30 eq 40 eq 50 eq

0

100

200

300

400

500

600

700

800

900

1000

Titration of Tb-1 (545 em) w/ cap-1

353 nm315 nm273 nm290 nm

Inte

nsity

(a.u

.)

Compare 290 nm exc. w/ 315 nm exc. over the cap1 concentration range

Cap1 w/ [Eu(1)]+

• Decrease in luminescence upon excitation at 270 nm, but corresponding increases at 317 nm and 365 nm

• Notice maximum increase at 317 nm occurs at 20 eq (compare to [Tb(1)]+

O

O

NH

O

N N

O

NHO

OOO

OHN

Eu3+

250 270 290 310 330 350 370 3900

10

20

30

40

50

60

70

80

90

100

Titration of Eu-1 (615 em) w/ cap-1

0 eq5 eq10 eq20 eq30 eq40 eq50 eq60 eq70 eq80 eq90 eq100 eq150 eq

Excitation wavelength (nm)

Inte

nsity

(a.u

.)

[Eu(1)]+ = 5.0 x 10-5 M

0 eq 5 eq 10 eq 20 eq 30 eq 40 eq 50 eq 60 eq 70 eq 80 eq 90 eq 100 eq 150 eq

0

10

20

30

40

50

60

70

80

90

Titration of Eu-1 (615 em) w/ cap-1

395 nm270 nm317 nm365 nm

Inte

nsity

(a.u

.)

Conclusion of initial experiments• The vanilloid structure is capable of sensitizing lanthanide

emissions• Simple capsaicinoids can be detected by EDTA-based

lanthanide chelates through easily observed luminescence signaling

• Ratiometric fluorescence detection is plausible• Methods are in place for characterizing the luminescent

response and nature of the complex(es) formed in solution• There remain some questions: How does cap1 bind to the

metal complex?

How does cap1 bind to [Ln(1)]+ ?

• Does cap1 bind through its two oxygen atoms and displace water molecules?

O

O

NH

O

cap1

• Luminescence lifetime titration– Compare rates of luminescence decay– A larger discrepancy between H2O and D2O means more

water molecules are coordinated

How does cap1 bind to [Ln(1)]+ ?

Small # of coordinated water molecules

Large # of coordinated water molecules

D2O H2O

• Luminescence lifetime titration– Compare rates of luminescence decay– A larger discrepancy between H2O and D2O means more

water molecules are coordinated

How does cap1 bind to [Ln(1)]+ ?

0 1 2 3 4 5 6 7 8 91

10

100

f(x) = 91.9195380816307 exp( − 1.07354625297813 x )R² = 0.999684130623913

f(x) = 94.2243082272687 exp( − 0.412333121124287 x )R² = 0.997728316408996

1 eq (290 nm)

D2OExponential (D2O)H2OExponential (H2O)

time (s)

Inte

nsity

(a.u

.)

Eu q (395) q (315)0eq 3.13 1.941eq 3.08 1.562eq 2.55 1.173eq 2.11 1.184eq 1.8 1.145eq 1.79 1.06

Tb q (290) q (315)0eq 2.55 2.951eq 2.99 2.872eq 3.05 2.8853eq 3.105 2.8754eq 3.15 2.885eq 3.16 2.92

How does cap1 bind to [Ln(1)]+ ?

• Cap1 does not appear to displace more than one water molecule, and only in the case of Eu

So exactly how does cap1 bind?

• Does cap1 bind the lanthanide through its two oxygens in a bidentate fashion?

How does cap1 bind to [Ln(1)]+ ?

220 240 260 280 300 320 340 360 380 400 4200

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

UV-Vis absorbance of [Tb(1)]+ w/ cap10 eq

0.2 eq

0.4 eq

0.6 eq

0.8 eq

1.0 eq

1.2 eq

1.4 eq

1.6 eq

1.8 eq

2.0 eq

3.0 eq

CAP 1 onlyWavelength (nm)

Abso

rban

ce

We would expect a shift in the absorbance wavelength of cap1 if this binding mode occurs!

O

HOO

HN

O

HOO

HN

+

+

How does cap1 bind to [Ln(1)]+ ?

• Must bind through some alternative way!

• How many molecules of cap1 can bind to one complex of [Ln(1)]+ ? (Job’s plot data)

How does cap1 bind to [Ln(1)]+ ?

• How can we figure this out? – Work with some other similar lanthanide

complexes and capsaicinoids– Obtain crystal structures of [Ln(1)]+ with cap1

How does cap1 bind to [Ln(1)]+ ?

Cap1 binding to naked Ln3+ ions

• Luminescence enhancement observed on the order of that observed in our chelates

• Evidence for partial removal of coordinated water molecules

H3CO

HO

NH

O

R

H2O

Ln3+ Ln3+

Designing new lanthanide complexes

• We want to be able to bind and detect the naturally-occurring capsaicinoids (all have a long alkyl chain)

H3CO

HO

NH

O

N

N

OHN

OO

O

O

O

HN

Ln3+

N

N

OHN

OO

O

O

O

HN

Ln3+

N

N

OHN

OO

O

O

O

HN

Ln3+

Synthetic pathwayO

NN

O

O

O

O

O

EDTA-BA

NN

OH

O

ONH

OH O

O

HN

NN

OH

O

ONH

OH O

O

HN

NH2

DMF DMF

Ln(OTf)3, 2NaOH

H2O Ln(OTf)3, 2NaOH

H2OLn= Eu or Tb

OTf=S

O

O-O CF3

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

NH222

[Ln(2)]+ [Ln(3)]+

2 more complexes

• Structural rigidity of aromatic ring system eases purification• [Ln(4)]+ is a precursor for expansion

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

[Ln(3)]+

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

[Ln(4)]+

NO2

O2N

Luminescence studies with [Tb(2)]+

• Luminescence augmentation is not as dramatic• Ligand 2 is an antenna (see intensity at 0 eq)

450 470 490 510 530 550 570 590 610 630 6500

50

100

150

200

250

300

350

Em @292 w/ [Tb(2)]+ and cap10eq

.2eq

.4eq

.6eq

.8eq

1eq

1.2eq

1.4eq

1.6eq

1.8eq

2.0eq

Wavelength(nm)

Inte

nsity

(A.U

.)

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

O

O

NH

O

cap1

• Excitation at 315 nm grows in (similar to [Tb(1)]+

Luminescence studies with [Tb(2)]+

230 250 270 290 310 330 350 3700

100

200

300

400

500

600

700

Exc @545 w/[Tb3+(L)] 0eq

.2eq

.4eq

.6eq

.8eq

1eq

1.2eq

1.4eq

1.6eq

1.8eq

2.0eq

3eq

4eq

5eq

6eq

7eq

8eq

9eq

10eqWavelength (nm)

Inte

nsity

(A.U

.)

N N

ONHO

OOOOHN

Ln3+

H2OOH2

OH2

O

O

NH

O

cap1

Future Work• Figure out what the heck is going on (really)• Chelate design must evolve!

– we currently focus on binding only one region of the capsaicin structure

– Incorporate functional groups on the chelate to hydrogen bond to the amide of capsaicinoids, and utilize the hydrophobic effect

– Synthesize and study other capsaicinoids and compare binding constants/stoichiometry

N N

O

NHO

O

R

OO

OHN

R

Ln3+

[Ln(#)]+

H3CO

HO

NH

O

R

Ultimate design

• Subject to change as we learn more!

H3CO

HO

NH

O

N

N

ONH

OO

O

O

O

HN

Ln3+

NHNH

S

NHNH

S

Acknowledgement

Longwood University- Department of Chemistry and Physics (research funding and start-up)- Cook-Cole fund, awarded January 2011