Chem 257 Lecture for Exp. 8, 2004

1. Exp. 7 Report: To be handed in this week, along with: • Relevant notebook pages • Vial containing triphenyl carbinol labeled with your notebook code, i.e., JST-I-38a

2. Exp. 8 Quiz. Be prepared for Quiz.

3. Required Reading. Lab Manual 8.1-10.

4. Relevant Material in Wade (5th): Ultraviolet spectroscopy 15-13 (p 666-672), 16-15 p(711-713), 18-5E (p 785-6). Friedel Crafts Alkylation: 17-11 (p 746-748) & p 980.

Exp. 8. and Chemiluminescence.

Purpose of the experiment:

1. Synthetic Organic Chemistry

• Acquaint you with Friedel-Crafts acylation chemistry

2. Physical Organic Chemistry

• Introduce you to photophysics

• Acquaint you with chemiluminescent reactions

1 Photochemical Terms and Principles.

Useful to consider a ground and energy diagram for an organic molecule.

Energy Level Diagram ISC S = singlet S T = triplet 1 A = Absorption F = Fluorescence T IC 1 P = A ISC = Intersystem crossing E F P IC = Internal Conversion ISC Absorption of a Emmission of a photon Vibrational energy loss So

Singlet State (S) • paired. • Ground state usually a singlet

(So).

• First excited singlet state = S1 So S1 • Nth excited singlet state = S n ground excited Triplet State (T) • Spin unpaired. • Lowest triplet state is usually an excited state (T1). • Nth excited triplet state = Tn T T Exceptions: 0 1 • Oxygen is a ground state Ground excited triplet! (O2)

2 Selection rules. • Spin allowed transitions do not involve a change in spin state

(Sn → Sm, Tn → Tm). • Thus ground singlet states are most easily excited to their first excited singlet states (So → S1). • Changing between spin states is called intersystem crossing and is spin forbidden and inefficient (Sn→ Tm , Tn → Sm)

A ISC P F, IC ISC S So S1 T1 o

Excited State Lifetimes • The lifetime refers to the amount of time that a particular state exists. • Excited singlet states of organic molecules (spin

allowed transition S1→So,) usually exist for on the order of nano (10-9 s) to microseconds (10-6 s)

• Excited triplet states (spin forbidden transition T1 -3 →So) can last for milliseconds (10 s) or more at room temperature.

A ISC P F, IC ISCIC S So S1 T1 o

3 Intersystem crossing (ISC). Crossing from one spin state to another, usually from the first ISC excited singlet state to the S1 lower energy first excited triplet state. Or T1 to So T1 Internal conversion (IC). E (Radiationless decay). A IC F Molecules in an excited ISC P state can go to a lower state of the same spin by loss of energy through S0 vibrational energy, and ultimately heat.

Fluorescence (F). • Molecules in their 1st excited singlet state can loose their energy by loss of a photon. ISC • Because the excited S1 singlet state usually has time to relax to its T1 ground vibrational state, E fluorescence emission A IC F maximum is usually at a ISCIC P longer wavelength than its absorption maximum. S0 • Very short lifetime because spin allowed transition, singlet to singlet).

4 Frank Condon Principle

• Excitation occurs faster S1 than a vibration (no change in geometry) S • Occurs from most E o hνA probable geometry of the ground state • Occurs from lowest vibrational state • Occurs from equilibrium bond geometry (center of potential curve) • Goes to vibrational level of the excited state with the highest probability of having Intensity the same geometry • Goes to a turning point Wavelength (edge of potential curve)

Frank Condon Principle S1 • Emmission occurs faster than a vibration (no change in geometry) E So

• Occurs between lowest hνE vibrational state of the excited electronic state • Occurs from equilibrium bond geometry (center of potential) • Goes to the vibrational state with the greatest probability of having same

geometry (turning point) Intensity

Wavelength

5 Frank Condon Principle S1 • Net result: Emmission occurs at a longer wavelength (lower E) than E So hνA absorption hνE • Emmission maximum is red-shifted compared to excitation • Thus lower wavelength (UV) that is invisible to the naked eye can be converted to visible light by the process of fluorescence Intensity

Wavelength

Color of Emitted Light. (source Louden, pg 661)

• Our eyes can only detect wavelenghts in the 400- 700 nm range (visible light)

• Sunlight at sea level consists of wavelengths from 290 nm and up

Range Type wavelength <200 vacuum UV 400-420 violet 200-280 UVC (germicidal) 420-491 280-320 UVB (tanning) 491-575 green 320-400 UVA (black light) 575-585 yellow 400-700 visible 585-647 orange 700- IR 647-700 red

6 Fluorescent Compounds for Home and Office Use • Optical brighteners (in clothes and computer paper) • Fluorescent compounds that absorb the <400 nm light in sunlight or fluorescent which are invisible to you • The absorbed light is re-emitted as visible light • Your clothes and term papers really are brighter!

HO N H - O3S N N N H N N H N NSON - 3 Blankophor B H N optical brightner OH

A Common Medicinal Fluorescent Compound • Quinine is an antimalarial alkaloid used to impart bitter taste to tonic water (gin and tonic a pleasing type of therapy) • Tonic water contains quinine which is fluorescent and will glow under black light (365 nm) • Black light derives its name because you cannot see it. • The blueish green glow from the excited quinine goes off instantly when the black light is turned off, as expected for fluorescence

OCH3 N absorption maximum 335 nm emission maxium 370 nm with a H tail out to 460 nm and a OH lifetime 10 nsec H N

7 Fluorescent Labels and Indicators for Biology • Fluorescein is used to label DNA and proteins • Fluorescein dipivalate is used as a probe of living cells

O esterases O lipases O Na + H2O O proteases O O CH3 CH OOO 3 Na OOO CH 3 CH3 CH3 CH3 fluorescein dipivalate basic form of fluorescein (not fluorescent) (fluorescent)

Membrane permeable Membrane inpermeable hydrolyzed in cell trapped inside cell

Green Fluorescent Protein (GFP) • of a protein leading to a fluorescent protein which emits green light • Requires oxygen • Used to tag proteins in cells by making fusion proteins and observing by fluorescence microsopy • Unsaturation shifts emmission wavelength to the visible

-H2O O -H2 -Phe-Ser-Tyr-Gly-Val- N Val69 N 64 69 O HO N H colorless Phe 64

GFP Chromophore

8 Phosphorescence (P). • Molecules in their 1st excited triplet state can loose their energy by loss of a photon. ISC • Because the excited S1 triplet state is usually lower than the singlet state, T1 phosphorescence E A maximum is usually much IC F ISCIC P longer than the fluorescence emission maximum. S0 • Very long lived because it is spin forbidden (triplet to singlet).

Phosphorescent Paints and Objects • Contain a phophorescent compound • Typically ZnS doped with Cu • First you must expose object to light to get a lot of the phosphorescent compounds into the excited state • The phophorescence does not go off instantaneously when the light is turned off, but can take minutes

9 Chemical Reaction. Excited states can also lead to bond dissociation, bond rearrangements, and reactions with other molecules.

Quantum Yield. • The quantum yield is a measure of efficiency of a particular event • Expressed as the fraction of the original photon absorbed that results in the particular event. • A quantum yield of 1 for a particular event, indicates that one of the particular event occurs for every photon absorbed.

Fluorescence Resonance Energy Transfer (FRET). • Exchange of excited state energy between two molecules through dipolar mechanism at close distances • Allowed, as there is no change in spin state • Acceptor (A) must have a lower energy excited state than the donor (D)

• Emitted light (FD) is of lower energy (lower frequency) than the light emitted from the donor (FA) • Used by biologists to detect interactions between biomolecules D D A D A A

A D FRET FA

FD

So S So S1 So S1 o

10 Use of FRET to detect specific mRNA sequence

In absence of complementary Donor Acceptor mRNA green emmission observed

In presence of FRET complementary mRNA FRET Donor Acceptor occurs and yellow emmission observed mRNA

Chemiluminescence. • Emission of a light as a result of a chemical reaction. • Occurs when the ground state of one molecule crosses with the excited state of a re-arranged or fragmented molecule. (Opposite of fluorescence where molecule goes from excited singlet state to ground state).

S1

∆G S0 hν

Reaction Coordinate

11 Common Commercial Use of Chemiluminescence. • Light sticks. Last 12 h at RT, days in freezer (not as bright). Cyalume by American Cyanamide. Quantum yield of 25%. • Labeling biomolecules

O O * Cl Cl O Cl O + H2O2 2 C O OO O O O Cl + Cl Cl * energy transfer Fluorescence

light

Bioluminescence • Emission of a photon as a result of a chemiluminescent reaction catalyzed by an in nature

Glowing bacteria V. harveyi bacteria glowing in colonizing a squid response to signals from E. coli (top) or salmonela (bottom)

12 - Luciferase System • Light emitting system of the • Requires an organic substrate luciferin, oxygen and the enzyme luciferase • Used commercially in non-isotopic biomolecule detection systems

O excite state Luciferase N N 2+ N N O* OH O2, Mg , ATP S S HO HO S S

luciferin + CO2 O N N O O HO S S

Exp. 8. Fluorescence and Chemiluminescence.

Goals of Your Experiment: • To synthesize a widely used fluorescent molecule, fluorescein by a Friedel Crafts acylation reaction • To examine the pH dependence of fluorescein fluorescence and correlate with structure • To study the chemiluminescent of , which is widely used in (criminology) • To use FRET (fluorescence resoance energy transfer) in the luminol chemiluminescent reaction to change the color of the emmitted light • To identify which of a number of samples may have contained

13 Synthesis of Fluorescein • Multistep reaction proceeding through many intermediates • Lewis acid catalyzed Friedel Crafts reaction (Wade 5th 17.11 p 747-8 & 980) 2+ •ZnCl2 is the Lewis acid (Zn ) • OH is o-, p-directing

OH O ZnCl 2 CO2H O + O OH HO O OH

O

O O O

HOO OH HO OH HO fluorescein OH

Experimental Procedures

• Preparation of anhydrous ZnCl2: The ZnCl2 is made anhydrous by heating in thermowell to drive off water (one does not want to hydrolyze the phthalic anhydride). • Friedel Crafts reaction: The reaction is conducted neat (without a solvent) at 180ºC for about 10 min in a test tube. • Side product: Bubbles which emanate at about 170ºC are due to water that is released in the reaction.

Note: Resorcinol is an irritant, heating ZnCl2 may liberate some HCl gas. Use a minifume hood.

• Workup. Product is freed from the inorganic materials

(ZnCl2) by trituration (dissolving away impurities from a solid) with hot acidic water. Fluorescein does not dissolve in acidic aqueous solutions, but the Zinc salts do.

14 pH Behavior of Fluorescein. • The product should visibly fluoresce in basic water, but not in acidic water • Only the basic form has extended conjugation • You will examine fluorescence of fluorescein at various pH's. O O

O Na + H2O O + 2 NaOH

HOO OH Na OOO

fluorescein aqueous base soluble form organic soluble form Non-fluorescent Fluorescent

Chemiluminescence of Luminol

• 1st step: Oxidation reaction (H2O2 or NaBO3 + catalyst). • 2nd step: Addition of peroxide. • 3rd Rxn: Intramolecular cyclization.

• 4th reaction: Retro Diels alder reaction to give N2 and light.

O O H Na N + 2 NaOH O + N2 (gas) N O + hv + 4 H O H + 2 H2O2 2 Na NH2 O NH2 O

+ H2O2 -2 H2O 4 1 Na O +2 NaOH O N + H2O2 O N N ON - 2 H O 2 Na NH O 2 2,3 NH2 O

15 Possible Mechanism for Excited State Formation • Retro Diels Alder Reaction • 4N + 2 electrons • Thermally allowed

Na

O O S1 Na O N O N + O N O hv Na N

NH2 O NH O Na 2

Catalysis • active metal salts, such as Cu2+ and Fe3+. You will use CuSO4 and which binds .

Driving force and source of excited state energy • Highly exothermic release of nitrogen • Exothermic acid-base reaction - formation of carboxylic acid salts

Emission characteristics • Maximum at 420 nm (excited state of 3-amino sodium phthalate • Emmission spectrum corresponds to the fluorescence spectrum of 3-aminophthalate

16 Chemiluminescence Energy Transfer • Reaction: FRET from 3-aminophthalate excited singlet state (donor) to fluorescein or rhodamine B (acceptors) • Reaction type: Through space energy transfer from higher energy excited singlet state of 3-nitrophthalate to the lower energy excited singlet state of fluorescein or rhodamine B • Driving force: Lower energy state of acceptor chromophore

Na O O O S1 O Na Na O N O FRET + O N O Na O OO NH2 O NH O Na 2 Na

Excited 3-aminophthalate can transfer energy to either rhodamine or fluorescein which emit different colored light

FRET FRET S1 S1 S 1 6.2 ns 420 nm lifetime 568 nm 530 nm

S 0 S0 S0 rhodamine B 3-aminophthalate fluorescein (basic form)

O O O O Na O Na O Na O Na O O OO N N CH3 NH O CH3 2 Na CH3 CH3

17 Use of the Luminol Reaction in Forensics heme found in • Used to detect presence of hemaglobin dried blood at crime scenes • Blood contains iron in the heme CH3 • The iron in dried blood can catalyze the oxidation of the H3C N N luminol (the first and rate Fe limiting step) H3CCHN N 3 • You will be asked to determine which of a number of stains contains blood by way of a luminol reaction. CO2H CO2H

Selected References

Application of and chemiluminescence in biomedical sciences. Methods Enzymol. 2000;305:333-45

Chemical generation of excited states: the basis of chemiluminescence and bioluminescence. Methods Enzymol. 2000;305:3-47

Luminescent Labels - More than Just an Alternative to Radioisotopes? Angew. Chemie Int. Ed. 1994, 33 1044-1072.

Electronic Structure of the Chromophore in Green Fluorescent Protein (GFP) J. Am. Chem. Soc., 1998:120 (36), 9370 -9371.

http://www.nemoto.co.jp/products/gss/index_e.html#example

18