Insight into the Zinc Sensing Mechanism of Zinpyr-1 and Application Toward Probe

Design

Christian R. GoldsmithMassachusetts Institute of Technology

2

Zinc in Biology

• Zn2+ a common cofactor in metalloproteins, may serve structural and/or catalytic roles

• Free or weakly bound Zn2+ found in diverse cells such as epithelial/myoepithelial, sperm, and pigment epithelial cells of retina

• Chelatable Zn2+ believed to have roles in regulating gene expression, apoptosis, enzyme function, and neurotransmission

Fluorescence image of an entire acute lumen section from a rat small intestine

stained with 10 M ZP3 for 20 min at 37 °C (Chang,

2003)

3

Zinc in Neurobiology

• Certain glutamatergic neurons in the hippocampus contain Zn2+ in vesicles in axon butons

• Zinc ion may be released into synaptic cleft upon neuronal excitation

• Zn2+ believed to decrease brain excitability through an allosteric interaction with NMDA receptor (Erreger, Traynelis, J. Physiol. 2005, 569, 381-393)

• Zn2+ a d10 metal ion- most sensors rely on change in fluorescence to detect metal ion

(Frederickson et al. The Neurochemist 2004, 10, 18-25)

4

Lead Compound: Zinpyr-1 (ZP1)

• 2’,7’-dichlorofluorescein (DCF) chosen for brightness and long wavelengths of excitation and emission

• Synthesized in one step from commercially available compounds

• Lone pairs on tertiary amines believed to quench fluorescence of the fluorescein

• Protons and metals bind to N atoms of ZP1, eliminating quenching process and restoring emission of fluorescein

Molecular Structure of ZP1(Walkup et al. J. Am. Chem. Soc., 2000, 122, 5644-5645)

OHO

Cl ClCOOH

N NN

N

N

N

O

5

ZinAlkylPyr (ZAP) 1-4

• Goal is to reduce affinity of probe for Zn2+

• Pyridylamine ligands attached via Mannich reaction

• Yields: 47% (ZAP1), 48% (ZAP2), 54% (ZAP3), 63% (ZAP4)

OHO

Cl ClCOOH

N N

R'

N

R'

NR R

ZAP1: R = R' = HZAP2: R = Me, R' = HZAP3: R = H, R' = PhZAP4: R = H, R' = C6F5

NH

NR+ (CH2O)n

DCFO

R'

Goldsmith and Lippard, Inorg. Chem. 2006, 45, 6474-6478

6

Photophysical Properties

• Methyl group on 6-position of pyridine lowers molar extinction coefficient and quantum yield, analogous to increasing methylation in MexZP1 series (Goldsmith and Lippard, Inorg. Chem. 2006, 45, 555-561)

• ZAP3 (high concentrations) and ZAP4 have much weaker absorption bands- deviations from Beer’s Law suggest possible aggregation

• Only ZAP4 displays enhanced fluorescence in the presence of Zn2+

Compound Absorption Emissionmax (nm) (M-1 cm-1) max (nm)

Apo-ZAP1 506 83000 525 0.82Apo-ZAP2 508 78000 527 0.74Apo-ZAP3 510 80300 (23700) 527 0.52Apo-ZAP4 514 9200 524 0.12Zn2-ZAP4 502 17000 520

0.56Measurements taken in 50 mM PIPES, 100 mM KCl, pH 7.0 buffer at 25° C;3.3 mM ZnCl2 was added to generate the samples of Zn2+-saturated ZAP4

7

Affinity of ZAP1-3 for Zn2+

• ZAP1-3 have no strong emissive response to Zn2+ at pH 7.0

• Large changes in optical spectrum upon Zn2+ titration-intensity of ~510 nm band decreases by ~30%

• Analysis of titrations (singular value decomposition) yield Kd values of:

10.2 (±1.4) M (ZAP1) 109 (±20) M (ZAP2)9.3 (±0.7) M (ZAP3)

• 6-Methyl group on ZAP2 weakens its affinity for Zn2+ 10-fold relative to that of ZAP1

Addition of ten 1.0 L aliquots and one 50 L aliquot of 10 mM ZnCl2

to a 5.5 M solution of ZAP1 in 100 mM KCl, 50 mM PIPES, pH

7.0 buffer at 25° C

8

pKa Values of ZAP1-3

• pKa values for emission turn-on during acidification:

8.30 (ZAP1)8.33 (ZAP2)7.88 (ZAP3)

• Curiously, values similar to that of ZP1 (8.4) which does have a fluorescent response to Zn2+ at pH 7.0

• ZAP1-3 display increased fluorescence in presence of Zn2+ at pH 9.0

• Maximal 2-fold turn-on for ZAP1 and ZAP2, 5-fold turn-on for ZAP3 at pH 9.0

Measurements performed on 0.5 M sample of dye in 100 mM KCl

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ZAP4 Zn2+-Response• ZAP4 a weaker base

than ZAP1-3 (pKa = 7.0)

• Unlike other sensors, 1 h required for full turn-on

• Fluorescence increase reversible with EDTA

0

5 105

1 106

1.5 106

2 106

2.5 106

520 540 560 580 600 620 640

Flu

ores

cenc

e In

tens

ity

Wavelength (nm)

A

B

C

A) 1.0 M ZAP4, B) 1.0 M ZAP4, 1.43 mM ZnCl2 t = 5 min,

C) 1.0 mM ZAP4, 1.43 mM ZnCl2 t = 1 h

2 106

2.2 106

2.4 106

2.6 106

2.8 106

3 106

3.2 106

0 500 1000 1500 2000 2500 3000F

luor

esce

nce

Inte

nsity

Time (s)

Reaction of ZAP4 with 0.77 mM ZnCl2

Red curve is single exponential fitGreen curve is double exponential fit

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ZAP4 Zn2+ Titration Analysis• First Kd (dissociation of 1:1

Zn2+: ZAP4 species) = 23 M

• Second Kd = 0.54 mM

• Both Kd values are near biologically relevant values

• Zn2-ZAP4 much more intense ( = 17000 M-1 cm-1) than ZAP4 or Zn-ZAP4, accounts for most of fluorimetric response to second equiv Zn2+

• First absorbance change of this magnitude in ZP-type compound

• Novel photophysical motif for Zn2+ sensing

0

5000

1 104

1.5 104

2 104

300 350 400 450 500 550 600 650 700

(M

-1 c

m-1

)

Wavelength (nm)

Zn2-ZAP4

Zn-ZAP4

ZAP4

Singular value decomposition analysis of ZAP4 spectrophotometric titration

with ZnCl2

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6-CO2H-ZAP4 Synthesis

OHO

Cl ClCO2H

N N

C6F5

N

C6F5

NNH

N+ (CH2O)n

6-CO2H-DCFO

C6F5

HO2C6-CO2H-ZAP4

• Synthesized as cell membrane impermeable version of ZAP4

• Yield: 58%

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6-CO2H-ZAP4 Characterization

0

0.2

0.4

0.6

0.8

1

2 4 6 8 10 12

Nor

mal

ize

Inte

grat

ed E

mis

sion

pH

6-CO2H-ZAP4

ZAP4

Compound Absorption Emissionmax (nm) (M-1 cm-1) max (nm)

Apo-ZAP4 514 9200 524 0.12Zn2-ZAP4 502 17000 520 0.56

Apo-CO2H-ZAP4 516 60000 527 0.14

Zn2-CO2H-ZAP4 504 52000 523 0.62

• Tertiary amine NH+ pKa value = 7.1

• First Kd of 22 (±4) M

• Absorption spectra of two compounds differ dramatically- no great change for 6-CO2H-ZAP4 with large excess of ZnCl2

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6-CO2H-ZAP4 Zn2+-Response

• 6-CO2H derivative has 5-fold maximal turn-on in emission during ZnCl2 titration- immediate

• Increased turn-on at mM levels of Zn2+ not observed- no second plateau as seen for ZAP4

• Best fit by A→B model

• Connection to more intense absorption?

Emission of a 0.5 M solution of 6-CO2H-ZAP4 with added ZnCl2

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Synaptic Vesicle Imaging• 6-CO2H-ZAP4 is cell

membrane impermeable

• Depolarize neurons in brain slice with KCl to promote endocytosis; dye from medium enters new vesicles

• Dye binds to Zn2+in vesicles

• Subsequent electrical excitation (tetanus) reduces dye emission

6-CO2H-ZAP4 TPEN Added Contrast Image

100 m

100 m

Merged Emission Image

2 m

Beforetetanus

After tetanus 1 m

Before After

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Conclusions

• Reducing the number of chelating atoms on the metal binding groups of ZP1 by one decreases binding affinity by four orders of magnitude

• ZAP probes can sense Zn2+ if the tertiary amines are sufficiently weak bases (ZAP4, 6-CO2H-ZAP4)

• ZAP4 has sizable enhancements in emission associated with each equiv of bound Zn2+; the second binding event is associated with an increase in the absorbance of the complex rather than an elevated quantum yield

• The 6-CO2H derivative of ZAP4 lacks the second emission increase associated with higher concentrations of Zn2+

• The 6-CO2H-ZAP4 dye can be used to access the Zn2+-containing synaptic vesicles in hippocampal neurons directly- strongest staining in region between DG and CA3

• Individual synapse imaged- loss of dye fluorescence may indicate loss of Zn2+ from vesicle

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AcknowledgementsPeople Funding

Prof. Stephen J. Lippard NIHDr. Ken-ichi OkamotoProf. Yasunori HayashiLippard Group

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ZAP4 Kinetics and Metal Sensitivity

• Reaction with first equiv Zn2+:kon = 1.13 (±0.05) ×

105 M-1 s-1

koff = 6.6 (±2.0) s-1

∆H‡ = 11.6 kcal mol-1

∆S‡ = 9.3 cal mol-1 K-1

• Metal sensitivity reflects faster rate of metal release- on par with Zinspy sensors (Nolan and Lippard. Inorg. Chem. 2004, 43, 8310-8317)

0

0.5

1

1.5

2

2.5

3

Ca2+

Mg2+

Mn2+

Fe2+

Co2+

Ni2+

Cu2+

Zn2+

Cd2+

F/F

o

Metal

100 M metal ion added (grey bar), followed by 100 M ZnCl2

(pink bar)

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ZP1 Zn2+-Response at pH 7.0

• Similar pKa values for tertiary amine NH+ species of ZAP1-3 and ZP1

• ZP1 has additional pyridine ring on each chelating group

• Rings implicated in quenching emission of apo-ZP1 at pH 7.0

• Could ZP1 bind H+ and Zn2+ in different modes?

• Missing pyridines in ZAP1-3 associated with elimination of fluorophore quenching

O

N

N

+HN

O

N

N

Zn2+

NH2O

Proton smaller, supports N2O coordination by ZP1, ZAP1-3

Zn2+ larger, supports N3O coordination by ZP1 as seen in crystal structure (Burdette et al. J. Am. Chem. Soc. 2001, 123, 7831-7841)

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Zinc Concentrations• Estimates of free Zn2+ concentrations in

most cell types range from femto to nanomolar

• Biological [Zn2+] upper limits: 10-30 M in synapse, low mM in vesicles

• Because of full d orbital, optical and magnetic based spectroscopies not useful for detection of Zn2+ in cells

• Major zinc probes (TSQ, Zinquin, ZPs) use change in fluorescence to signal presence of Zn2+

• Most zinc probes have Kds ~ 1 nM

• Other sensors (TSQ, Zinquin) bind zinc in complicated manner

• Non-ratiometric sensors cannot measure [Zn2+] by themselves, only detect its presence

Compound Kd (nM)

ZnAF-1 0.78ZnAF-2 2.7ZnAF-1F 2.2ZnAF-2F 5.5Zinbo-5 2.2ZP1 0.7ZP2 0.5

Select Zn2+ sensors from Nagano,

O’Halloran, and Lippard