detecting ‘heat’ with light: luminescent sensors for capsaicin
DESCRIPTION
Detecting ‘Heat’ with Light: Luminescent Sensors for Capsaicin. Christopher G. Gulgas. Chemistry review: Hydrogen bonding. Molecules with O-H bonds or N-H bonds can “hydrogen bond” to other molecules. δ -. δ +. δ +. Chemistry review: Hydrogen bonding. δ -. δ -. Chemistry review: Lipids. - PowerPoint PPT PresentationTRANSCRIPT
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