Hermanson G. Bioconjugate Techniques, Second Edition

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440 9. Fluorescent Probes

2. Dissolve sodium periodate in water to a ﬁ nal concentration of 100 mM. Protect from

light. Add 0.1 ml of this stock periodate solution to each ml of the antibody solution.

3. React for 15 minutes at room temperature, protected from light.

4. Quench the reaction by addition of 0.1 ml of glycerol per ml of reaction volume, mix, and

then react for an additional 15 minutes. Remove excess reagents by gel ﬁ ltration using a

desalting column, and performing the chromatography using the phosphate buffer.

5. Adjust the concentration of IgG in the puriﬁ ed preparation to 1 mg/ml by the addition of

0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5.

Modiﬁ cation of Oxidized IgG with AMCA-Hydrazide

1. Dissolve AMCA-hydrazide in DMF at a concentration of 0.4 mg/ml. Protect from light.

2. Add 50–100 l of the AMCA-hydrazide stock solution to each ml of the oxidized anti-

body solution.

Note: At a level of 50- l probe addition, polyclonal human IgG will be modiﬁ ed at a

level that gives an F / P ratio of about 0.113. Since the labeling occurs only at the oxidized

carbohydrate sites, the ﬂ uorophore incorporation typically is less than that observed

when using amine-reactive probes.

3. React for 30 minutes at room temperature in the dark.

4. Remove excess ﬂ uorophore by dialysis or gel ﬁ ltration using a desalting resin. Protect the

labeled immunoglobulin from light.

Determine the F / P ratio by measuring the absorbance at 345 and 280 nm.

4. BODIPY Derivatives

BODIPY ﬂ uorophores are a class of probes based on the fused, multi-ring structure, 4,4-

diﬂ uoro-4-bora-3a,4a-diaza- s-indacene ( Figure 9.27 ) (Invitrogen) (U.S. patent 4,774,339). This

fundamental molecule can be modiﬁ ed, particularly at its 1, 3, 5, 7, and 8 carbon positions, to

produce new ﬂ uorophores with different characteristics. The modiﬁ cations cause spectral shifts

in its excitation and emission wavelengths, and can provide sites for chemical coupling to label

biomolecules.

The BODIPY derivatives typically have high extinction coefﬁ cients and excellent QY, often

greater than 0.8. Their spectral characteristics are relatively insensitive to changes in pH.

Figure 9.27 The basic structure of BODIPY ﬂ uorophores.

Luminescent changes with shifts in pH usually are due to reconﬁ guration of a ﬂ uorophore ’s

-electron cloud if an atom on the ring system becomes protonated or unprotonated. Since the

BODIPY structure lacks an ionizable group, alterations in pH have no effect on its spectral

attributes.

The emission spectra of BODIPY derivatives normally display narrow bandwidths, providing

intensely ﬂ uorescent labels for biomolecules. Unfortunately, they also have very small Stoke ’s

shifts, typically on the order of only 10–20 nm. Excitation at the optimal wavelength may

cause some interference in measurements at the emission wavelength due to light scattering or

cross-over from the wide bandwidth of the excitation source. The dyes usually require excita-

tion at sub-optimal wavelengths to prevent this problem.

The following sections discuss the major BODIPY derivatives that are reactive toward par-

ticular functional groups in proteins and other molecules.

Amine-Reactive BODIPY Derivatives

A number of BODIPY derivatives that contain reactive groups able to couple with amine-

containing molecules are commonly available. The derivatives either contain a carboxy-

late group, which can be reacted with an amine in the presence of a carbodiimide to create

an amide bond, or an NHS ester derivative of the carboxylate, which can react directly with

amines to form amide linkages. The three discussed in this section are representative of this

amine-reactive BODIPY family. The two NHS ester derivatives react under alkaline conditions

with primary amines in molecular targets to form stable, highly ﬂ uorescent derivatives. The

carboxylate derivative can be coupled to an amine using the EDC/sulfo-NHS reaction discussed

in Chapter 3, Section 1.2.

The only disadvantage of using BODIPY ﬂ uorophores to label amines in macromolecules is

the tendency for ﬂ uorescence quenching to occur if multiple sites on one molecule are modiﬁ ed.

Especially with proteins, using an amine-reactive probe usually results in a number of sites

being modiﬁ ed on each molecule. All ﬂ uorophores experience some quenching effect if the

degree of substitution is high, because probe–probe interactions are possible that can transfer

energy from an excited-state ﬂ uorophore to a ground-state ﬂ uorophore before luminescence

occurs. BODIPY probes, however, are especially notorious for probe–probe quenching effects.

For this reason, the manufacturer (Invitrogen) recommends that the amine-reactive BODIPY

probes only be used to modify substances that have the potential for just one substitution per

molecule. In this sense, BODIPY ﬂ uorophores are particularly well suited for tagging DNA

probes at the 5  end or lipid molecules on their head groups. Oligonucleotides modiﬁ ed to

contain an amine on their 5  phosphate group (Chapter 27, Section 2.1) are particularly good

candidates for labeling with this ﬂ uorophore. Other BODIPY probes that contain reactivity

toward non-amine functionalities such as sulfhydryls or polysaccharides may be more effective

at labeling macromolecules like proteins, since these groups occur at more limited sites within

the molecules and the modiﬁ cation level can be better controlled.

BODIPY FL C

-SE

BODIPY FL C

-SE is 4,4-diﬂ uoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-propionic

acid, succinimidyl ester (Invitrogen). The derivatization to the base BODIPY molecule

4. BODIPY Derivatives 441

442 9. Fluorescent Probes

results in ﬂ uorescent properties which mimic ﬂ uorescein in its emission wavelength. The mol-

ecule thus emits light in the green region of the spectrum. The NHS ester on its propionic

side chain provides amine reactivity, resulting in amide bond linkages with modiﬁ ed molecules

(Figure 9.28 ).

This ﬂ uorophore has an excitation maximum at 502 nm and an emission maximum at

510 nm. The small Stoke ’s shift of only 8 nm creates some difﬁ culty in discrete excitation with-

out contaminating the emission measurement with scattered or overlapping light. The extinc-

tion coefﬁ cient of the molecule in methanol is about 77,000 M

 1

at 502 nm.

BODIPY FL C

-SE is insoluble in aqueous solution, but may be dissolved in DMF or DMSO

as a concentrated stock solution prior to addition of a small aliquot to a reaction. For aqueous

reactions, a pH range of 7–9 is optimal. Avoid amine-containing buffers. The reaction also may

be done in organic solvent.

Since BODIPY ﬂ uorophores are easily quenched if substitutions on a molecule exceed a 1:1

stoichiometry, modiﬁ cation of proteins with this ﬂ uorophore probably won ’t yield satisfactory

Figure 9.28 The side-chain NHS ester of this BODIPY derivative can be used to modify amine-containing mol-

ecules, forming amide bond linkages.

results. However, for labeling molecules which contain only one amine group, BODIPY FL

-SE will give intensely ﬂ uorescent derivatives.

BODIPY 530/550 C

is 4,4-diﬂ uoro-5,7-diphenyl-4-bora-3a,4a-diaza- s -indacene-3-propionic

acid (Invitrogen). This derivative of the basic BODIPY structure contains two phenyl rings off

the No. 5 and 7 carbon atoms and a propionic acid group on the No. 3 carbon atom. The

carboxylate group may be used to attach the ﬂ uorophore to amine-containing molecules via a

carbodiimide reaction to create an amide bond. The substituents on this BODIPY ﬂ uorophore

result in alterations to its spectral properties, pushing its excitation and emission maximums up

to higher wavelengths.

The excitation maximum for the molecule occurs at 535 nm and its emission at 552 nm.

Its Stoke ’s shift is slightly greater than some of the other BODIPY ﬂ uorophores, producing a

17 nm separation between excitation and emission peaks. BODIPY 530/550 C

has an extinc-

tion coefﬁ cient in methanol of about 62,000 M

 1

at 535 nm.

BODIPY 530/550 C

is insoluble in aqueous solution, but it may be dissolved in DMF or

DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction.

Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction

as discussed in Chapter 3, Section 1.2 ( Figure 9.29 ). However, modiﬁ cation of proteins with

this ﬂ uorophore probably won ’t yield satisfactory results, since BODIPY ﬂ uorophores are eas-

ily quenched if substitutions on a molecule exceed a 1:1 stoichiometry. For labeling molecules

which contain only one amine group, such as DNA probes modiﬁ ed at the 5  end to contain an

amine (Chapter 27, Section 2.1), BODIPY 530/550 C

will give intensely ﬂ uorescent derivatives.

BODIPY 530/550 C

-SE

BODIPY 530/550 C

-SE is 4,4-diﬂ uoro-5,7-diphenyl-4-bora-3a,4a-diaza- s-indacene-3-propionic

acid, succinimidyl ester (Invitrogen). The compound is an analog of BODIPY 530/550 C

that

4. BODIPY Derivatives 443

444 9. Fluorescent Probes

contains an active NHS ester on its propionic acid side chain (Chapter 2, Section 1.4). The ester

reacts with primary amines to form stable amide bonds.

The excitation maximum for BODIPY 530/550 C

-SE occurs at 533 nm and its emission at

550 nm. Its Stoke ’s shift is relatively small and may not be enough to avoid completely prob-

lems of excitation-light interference in emission measurements. The molecule has an extinction

coefﬁ cient in methanol of about 70,000 M

 1

at 533 nm.

BODIPY 530/550 C

-SE is insoluble in aqueous solution, but may be dissolved in DMF

or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction.

Coupling to amine-containing molecules proceeds by nucleophilic attack at the carbonyl group,

release of the NHS leaving group, and formation of an amide linkage ( Figure 9.30 ). The reac-

tion may be done in buffered environments having a pH range of 7–9. However, modiﬁ cation

of proteins with this ﬂ uorophore may not yield satisfactory results, since BODIPY ﬂ uorophores

are easily quenched if substitutions on a molecule result in a high molar ratio of incorporation.

For labeling molecules which contain only one amine group, such as DNA probes modiﬁ ed at

the 5  end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C

-SE will give

intensely ﬂ uorescent derivatives.

Aldehyde/Ketone-Reactive BODIPY Derivatives

Hydrazide groups react with aldehyde and ketone groups to form hydrazone linkages (Chapter 2,

Section 5.1). Three BODIPY derivatives are available that contain a hydrazine group modiﬁ cation

of carboxylate side chains. Biomolecules such as proteins that don ’t normally possess aldehyde

residues can be modiﬁ ed to contain them by a number of chemical means (Chapter 1, Section 4.4).

In addition, DNA and RNA may be modiﬁ ed with hydrazide-containing ﬂ uorophores by

a transamination reaction of their cytosine residues using bisulﬁ te as a catalyst (Chapter 27,

Section 2.1) (Draper and Gold, 1980).

Figure 9.29 This BODIPY ﬂ uorophore contains a carboxylate group that can be attached to amine-containing

molecules using a carbodiimide reaction.

BODIPY 530/550 C

Hydrazide

BODIPY 530/550 C

hydrazide is 4,4-diﬂ uoro-5,7-diphenyl-4-bora-3a,4a-diaza- s -indacene-3-

propionyl hydrazide, a derivative of the basic BODIPY structure that contains two phenyl rings

off the No. 5 and 7 carbon atoms and a propionic acid hydrazide group on the No. 3 carbon

Figure 9.30 The NHS ester group of this BODIPY compound provides amine reactivity.

4. BODIPY Derivatives 445

446 9. Fluorescent Probes

atom (Invitrogen). The hydrazide group reacts with aldehyde- or ketone-containing molecules

to form hydrazone linkages ( Figure 9.31 ). The compound may be used to label glycoproteins

or other carbohydrate-containing molecules after oxidation of their polysaccharide portions

with sodium periodate to yield aldehydes.

The excitation maximum for BODIPY 530/550 C

hydrazide occurs at 534 nm and

its emission at 551 nm. The molecule has an extinction coefﬁ cient in methanol of about

79,000 M

 1

at 534 nm.

BODIPY 530/550 C

hydrazide is insoluble in aqueous solution, but may be dissolved in

DMF or methanol as a concentrated stock solution prior to addition of a small aliquot to a

reaction. Coupling to aldehyde-containing molecules occurs rapidly with the formation of a

hydrazone linkage. The reaction may be done in buffered environments having a pH range of

5–10. However, modiﬁ cation of glycoproteins with this ﬂ uorophore may not yield satisfactory

Figure 9.31 The side-chain hydrazide group of this BODIPY derivative can be used to label aldehyde-containing

molecules. Glycoconjugates may be labeled after oxidation of carbohydrates with sodium periodate to produce

the required aldehydes.

results, since BODIPY ﬂ uorophores are easily quenched if substitutions on a molecule result in

a high molar ratio of incorporation.

BODIPY 493/503 C

Hydrazide

BODIPY 493/503 C

hydrazide is 4,4-diﬂ uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza- s-indacene-

8-propionyl hydrazide (Invitrogen). Unlike BODIPY 530/550 C

hydrazide, this BODIPY derivative

contains substituents that shift to lower wavelengths the spectral characteristics of its ﬂ uorescence.

The molecule is highly reactive toward aldehyde-containing compounds, including glycoproteins

which have been oxidized with sodium periodate to create the requisite groups ( Figure 9.32 ).

The excitation maximum for BODIPY 493/503 C

hydrazide occurs at 498 nm and its emis-

sion at 506 nm. Since this is an extremely small Stoke ’s shift, it may be difﬁ cult to avoid com-

pletely problems of excitation-light scattering interference in critical emission measurements

unless sub-optimal excitation wavelengths are used. The molecule has an extinction coefﬁ cient

in methanol of about 92,000 M

 1

at 493 nm.

Figure 9.32 Reaction of this BODIPY ﬂ uorophore with aldehyde groups creates hydrazone linkages.

4. BODIPY Derivatives 447

448 9. Fluorescent Probes

BODIPY 493/503 C

hydrazide is insoluble in aqueous solution, but may be dissolved in

DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reac-

tion mixture. Coupling to aldehyde-containing molecules occurs rapidly with the formation of

a hydrazone linkage. The reaction may be done in buffered environments having a pH range of

5–10. However, modiﬁ cation of glycoproteins with this ﬂ uorophore may not yield satisfactory

results, since BODIPY ﬂ uorophores are easily quenched if substitutions on a molecule result in

a high molar ratio of incorporation. Limiting the modiﬁ cation level by reacting no more than a

2- to 4-fold molar excess of probe to the amount of glycoconjugate present may overcome this

quenching problem.

BODIPY FL C

Hydrazide

BODIPY FL C

hydrazide is 4,4-diﬂ uoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene-3-propionyl

hydrazide (Invitrogen). Unlike the two BODIPY hydrazide derivatives discussed above, this

derivative contains substituents that produce luminescent characteristics similar to that of ﬂ uo-

rescein, particularly with regard to ﬂ uorescing in the green region of spectrum. The molecule

is highly reactive toward aldehyde-containing compounds, including glycoproteins which have

been oxidized with sodium periodate to create the requisite aldehyde groups ( Figure 9.33 ).

Figure 9.33 Modiﬁ cation of aldehyde-containing molecules can be done through this BODIPY derivative ’s

hydrazide group.

The excitation maximum for BODIPY FL C

hydrazide occurs at 503 nm and its emission at

510 nm. The extremely small Stoke ’s shift makes it difﬁ cult to avoid problems of excitation-light

interference in critical emission measurements unless sub-optimal excitation wavelengths are used.

The molecule has an extinction coefﬁ cient in methanol of about 71,000 M

 1

at 503 nm.

BODIPY FL C

hydrazide is insoluble in aqueous solution, but may be dissolved in DMF

or methanol as a concentrated stock solution prior to addition of a small aliquot to a reaction

mixture. Coupling to aldehyde-containing molecules occurs rapidly with the formation of a

hydrazone linkage. The reaction may be done in buffered environments having a pH range of

5–10. However, modiﬁ cation of glycoproteins with this ﬂ uorophore may result in ﬂ uorescent

quenching effects if substitutions on a molecule are at a high molar ratio of incorporation.

Limiting the modiﬁ cation level by reacting no more than a 2- to 4-fold molar excess of probe

to the amount of glycoconjugate present may help to overcome the quenching problem.

Sulfhydryl-Reactive BODIPY Derivatives

Three BODIPY derivatives are available for labeling sulfhydryl-containing molecules. The

ability to label  SH groups in proteins with sulfhydryl-reactive probes provides a means of

directing the modiﬁ cation reaction to a more limited number of sites than occurs when using

amine-reactive chemistries. Directed coupling potentially can avoid active centers or bind-

ing regions. The ﬁ rst two sulfhydryl-reactive probes discussed in this section make use of

iodoacetyl derivatives off the basic BODIPY molecule. The third probe is a bromomethyl deriv-

ative that also has good reactivity toward sulfhydryls.

BODIPY FL IA

BODIPY FL IA is N-(4,4-diﬂ uoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene-3-propionyl)-N  -

iodoacetylethylenediamine, an intensely ﬂ uorescent derivative of the basic BODIPY structure

which is useful in modifying sulfhydryl groups (Invitrogen). The iodoacetyl group reacts with  SH

groups in proteins and other molecules to form a stable thioether linkage ( Figure 9.34 ). The

reactive group is at the end of a reasonably long spacer arm, providing enough length to avoid

steric problems in modifying sulfhydryls not easily accessible at the surface of macromolecules.

4. BODIPY Derivatives 449