CHAPTER 1 CHAPTER 3 Fluorophores and Cli ck Chemistry ... · CHAPTER 3 ck Chemistry and Molecular...

CHAPTER 3

ck Chemistry and

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CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes® HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

TH

REE

CHAPTER 3

Click Chemistry and Other Functional Group Modi�cations

3.1 Click Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Principles of Bioorthogonal Labeling and Click Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Click-iT® Azide and Alkyne Labeling Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Azide- and Alkyne-Derivatized Dyes and Biotinylation Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Azide- and Alkyne-Modi�ed Nucleosides and Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Azide- and Alkyne-Modi�ed Monosaccharides, Fatty Acids and Isoprenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Heterobifunctional Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Click-iT® Reaction Bu�ers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Click-iT® Tools for Cell Proliferation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Click-iT® EdU Flow Cytometry Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Click-iT® EdU Imaging Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Click-iT® EdU HCS Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Click-iT® EdU Microplate Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Click-iT® TUNEL Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Click-iT® Tools for Detecting RNA and Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Click-iT® RNA Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Click-iT® Nascent Protein Synthesis Detection Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Data Table 3.1 Click Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Product List 3.1 Click Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

3.2 Reagents for Modifying Alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Alcohols in Proteins: Serine, Threonine and Tyrosine Residues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Serine and Threonine Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Tyrosine Residues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Alcohols in Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Dichlorotriazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

N-Methylisatoic Anhydride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Dansyl Aminophenylboronic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Alcohol Modi�cation in Organic Solvents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Isocyanates Prepared from Acyl Azides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Acyl Nitriles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Data Table 3.2 Reagents for Modifying Alcohols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Product List 3.2 Reagents for Modifying Alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

3.3 Reagents for Modifying Aldehydes and Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Introducing Aldehydes and Ketones into Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Periodate Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Galactose Oxidase–Mediated Oxidation and Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Coupling Hydrazines and Amines to Amine-Containing Biomolecules without Introducing Aldehydes and Ketones . . . . . . . . . . . . . . . . . . 139

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IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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Chapter 3 — Click Chemistry and Other Functional Group Modi�cations

Introducing a Hydroxylamine into a Biomolecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Hydrazines and Hydroxylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Reactivity of Hydrazine and Hydroxylamine Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Polar Fluorescent Hydrazides and Hydroxylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

NBD Methylhydrazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Biotin Hydrazides and Biotin Hydroxylamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Aliphatic and Aromatic Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Data Table 3.3 Reagents for Modifying Aldehydes and Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Product List 3.3 Reagents for Modifying Aldehydes and Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

3.4 Derivatization Reagents for Carboxylic Acids and Carboxamides . . . . . . . . . . . . . . . . . . . . . . 145

Coupling Hydrazines, Hydroxylamines and Amines to Carboxylic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Modi�cation in Aqueous Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Modi�cation in Organic Solvents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Hydrazine, Hydroxylamine and Aliphatic Amine Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Enzyme-Catalyzed Transamidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Esteri�cation of Carboxylic Acids with Fluorescent Diazoalkanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Fluorescent Alkyl Halides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Fluorescent Tri�uoromethanesulfonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

4-Sulfo-2,3,5,6-Tetra�uorophenol (STP) and N-Hydroxysulfosuccinimide (NHSS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

Data Table 3.4 Derivatization Reagents for Carboxylic Acids and Carboxamides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Product List 3.4 Derivatization Reagents for Carboxylic Acids and Carboxamides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 3.1 Click Chemistry

�e amine– and thiol-reactive labeling chemistries described in Chapter 1 and Chapter 2 are generally used in one of two ways: (1) labeling of puri�ed proteins or other biopolymers yielding conjugates that are subsequently applied to cell or tissue specimens, or (2) non-selective in situ labeling of total cellular thiol or amine content. In situ labeling of speci�c molecular populations—such as proteins and nucleic acids that have been newly synthesized in some experimental time window of interest—is not feasible due to the ubiquitous distribu-tion of amines and thiols in cells, as well as in the media in which they are maintained. Click-iT® labeling technology overcomes this obstacle

3.1 Click ChemistryPrinciples of Bioorthogonal Labeling and Click Chemistry

Figure 3.1.1 Click-iT® copper-catalyzed azide–alkyne cycloaddition chemistry applied to detection of A) nucleic acids, B) proteins, C) carbohydrates and D) lipids. The reaction partners are A) 5-ethynyl-2’-deoxyuridine (EdU) and Alexa Fluor® 488 azide, B) L-homopropargylglycine (HPG) and Alexa Fluor® 488 azide, C) N-azidoacetylgalactosamine and Alexa Fluor® 488 alkyne and D) 15-azidopentadecanoic acid and Alexa Fluor® 488 alkyne (D). In each case, the left-hand partner is a metabolic precursor that can be incorporated into proteins and nucleic acids via de novo synthesis or post-translational modi�cation pathways.

CH2NH

NN

N

NH2 O NH2

C

O

O

SO3SO3

C

O

(CH) 14CHO

O

Cu+

NH2 O NH2

C

O

O

SO3SO3

CCCH2NH

O

CH

(CH2)14NC N NHO

O

Cu

+O

HONH

CH2

OH

CCH2N N N

O

OHHO

NH2 O NH2

C

O

O

SO3SO3

CCCH2NH

O

CHO

HONH

CH2

OH

CCH2

O

OHHO

NH2 O NH2

C

O

O

SO3SO3

CCH2NH

O

NN

N

Cu

NH2 O NH2

C

O

O

SO3SO3

CNN (CH2)6NHN

O

+CH

NH2

CHO

O

CH2CH2C CH

CH2CH2

NH2 O NH2

C

O

O

SO3SO3

C(CH2)6NH

O

NN

NCHC

NH2

HO

O

N

O

OH

HOCH2

HN

O

O

NH2 O NH2

C

O

O

SO3SO3

C(CH2)6NH

O

NN

N

CuN

O

OH

HOCH2

HN

O

O

C CH NH2 O NH2

C

O

O

SO3SO3

CNN (CH2)6NHN

O

+A

B

C

D

by employing bioorthogonal reactive chemistry, in which the reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms.1–3 In addition to reaction selectivity, in situ labeling methods should allow reactivity under mild conditions and in predominantly aqueous solvent conditions.

Although several known chemistries ful�ll the requirements de-scribed above,1,3 Click-iT® labeling technology is founded upon one of the most successful and versatile bioorthogonal labeling reactions cur-rently available—the copper-catalyzed azide–alkyne cylo addition 4,5 (Figure 3.1.1). Application of this reaction to in situ labeling of cells



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is a two-step process. First, one reaction partner—either an azide or alkyne linked to a “building block” such as a nucleotide, nucleoside, amino acid, monosaccharide or fatty acid—is biosynthetically incor-porated. Subsequently, the other reaction partner—the complementary alkyne or azide linked to a �uorescent dye, biotin or other detection reagent—is “clicked” into place in the presence of catalytic copper (I). One reaction partner must be an azide derivative and the other an alkyne derivative, but either functional moiety can serve as the bio-synthetically incorporated molecule or the detection molecule 6 (e.g., L-azidohomoalanine (AHA) + Alexa Fluor® 488 alkyne is the inverse of the reaction scheme shown in Figure 3.1.1B).

Another important aspect of the azide and alkyne reaction part-ners is their small size (Figure 3.1.2). Expression tags such as Green Fluorescent Protein (GFP) provide the ultimate in labeling speci�city be-cause their linkage to proteins of interest is genetically prescribed. Once the GFP transgene has been inserted into a cell (BacMam Gene Delivery

Figure 3.1.2 Relative size of detection molecules commonly used in cellular analysis. Because the azide and alkyne moieties can be used interchangeably to optimize labeling con�gurations, R1 can be either the biomolecule of interest or the detection reagent. For biotin and Alexa Fluor® 488, R2 represents the biomolecule of interest.

+

C

H2N O

O

NH2

SO3 SO3

O

R2O C

O

–

––

R1 NR1 NN

Isotope(s)

MW

Click-iT® alkyne Click-iT® azide Biotin Alexa Fluor® 488 Streptavidin IgG antibody

3 25 42 ~300 500 ~68,000 ~150,000

Both required for detection Both required for detection

R2

Table 3.1 Molecular Probes® azide and alkyne derivatives.

Compound or Fluorophore Azide or Alkyne Cat. No. Application

Azide- or Alkyne-Modi�ed Biomolecules for Metabolic or Enzymatic Labeling

Click-iT® AHA (L-azidohomoalanine) azide C10102 Monitor nascent protein synthesis or inhibition

Click-iT® HPG (L-homopropargylglycine) alkyne C10186

Click-iT® farnesyl alcohol, azide azide C10248 Identi�cation of isoprenylated proteins

Click-iT® geranylgeranyl alcohol, azide azide C10249

Click-iT® fucose alkyne (tetraacetylfucose alkyne) alkyne C10264 Identi�cation of fucoslyated proteins

Click-iT® palmitic acid, azide azide C10265 Identi�cation of protein fatty acylation

Click-iT® myristic acid, azide azide C10268

Click-iT® GalNAz (tetraacetylated N-azidoacetylgalactosamine) azide C33365 Identi�cation of O-linked glycoproteins, including O-GlcNAc

Click-iT® ManNAz (tetraacetylated N-azidoacetylmannosamine) azide C33366 Identi�cation of sialic acid–modi�ed glycoproteins

Click-iT® ManNAz (tetraacetylated N-azidoacetylglucosamine) azide C33367 Identi�cation of O-GlcNAc–modi�ed glycoproteins

Azide- or Alkyne-Modi�ed Fluorophores and Haptens for Detection (Ex/Em) *

Alexa Fluor® 488 (495/519) azidealkyne

A10266A10267

Fluorescent dye or hapten †


A20012A20013

Fluorescent dye


A10270A10275

Fluorescent dye

Alexa Fluor® 647 (650/668) azide A10277 Fluorescent dye

Biotin (NA)‡ azidealkyne

B10184B10185

Hapten

Oregon Green® 488 (496/524) azidealkyne

O10180O10181


Tetramethylrhodamine (TAMRA) (555/580) azidealkyne

T10182T10183


* Absorption and �uorescence emission maxima, in nm. † As hapten, use with anti-dye antibody; see Section 7.4. ‡ Requires streptavidin for detection; see Section 7.6.

and Expression Technology—Note 11.1), in situ labeling is obtained without any outside intervention using the cellular transcription and translation machinery. However, the �nite size of GFP (~27,000 dal-tons) sometimes causes functional perturbations and has spurred the development of alternative, smaller-sized expression tags such as the TC-FlAsH™ tetracysteine tag and biarsenical ligand system (T34561, T34562, T34563; Section 2.2). Furthermore, nucleic acids, lipids, gly-cans and post-translational protein modi�cations can only be detected indirectly by genetically encoded protein reporters. �e small size of alkyne and azide tags allows the biosynthetic building blocks to which they are attached to be processed by enzymes, such as nucleotide poly-merases and aminoacyl tRNA synthetases, that have poor tolerance for substrates with larger modi�cations such as �uorescent organic dyes.7

�e 1,2,3-triazole linkage between the azide and alkyne reac-tion partners (Figure 3.1.1) is extremely stable. It is not susceptible to hydrolysis, oxidation or reduction, and it survives ionization in mass


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofi sher.com/probes





spectrometry (MS) analysis. �e reaction is also regiospeci�c, yielding exclusively 1,4-disubsti-tuted-1,2,3-triazole linkages (Figure 3.1.1). �e copper (I) catalyst is both an essential feature of the reaction and its most problematic aspect in terms of applications.1,3,8 Without the copper (I) catalyst, which accelerates the rate of the reaction by a factor of >106, the reaction is impractically slow. For convenience, copper (I) is usually prepared in situ by reduction of extraneously added copper (II) using ascorbate or TCEP (T2556, Section 2.1). Insu�cient reductive capacity can result in attenuation of in situ reactions in highly oxidizing environments.9 Copper is cytotoxic, due at least in part to its capacity for sensitizing oxidative damage to proteins and nucleic acids, and limit-ing applications of the azide–alkyne cyloaddition reaction in live cells. Copper/ascorbate treatment also causes extinction of R-phycoerythrin (R-PE) and GFP �uorescence.10 An excellent analysis of these considerations, together with a list of practical recommendations for their management with respect to bioconjugation applications of copper-catalyzed azide–alkyne cyloaddition chemistry, has been published by Finn and co-workers.8 Our Click-iT® product portfolio, consisting of indi-vidual azide and alkyne labeling reagents and application-speci�c kits, is described in detail below.

Click-iT® Azide and Alkyne Labeling ReagentsAlthough in situ labeling of biomolecules for cytochemical and proteomic analysis is perhaps

the most notable application of Click-iT® technology, it is by no means the only one. �is speci�c and direct labeling methodology can also be applied to bioconjugate preparation,8 surface and particle functionalization and molecular ligations. Our Click-iT® azide and alkyne labeling re-agents support these applications and also provide foundational tools for developing new in situ labeling applications (Table 3.1).

Azide- and Alkyne-Derivatized Dyes and Biotinylation ReagentsWe o�er a rich selection of azide- and alkyne-derivatized �uorescent dyes for coupling to

complementary azide- and alkyne-functionalized biomolecules:

• Alexa Fluor® 488 azide (A10266, Figure 3.1.1) and alkyne (A10267, Figure 3.1.1)• Alexa Fluor® 555 azide (A20012) and alkyne (A20013)• Alexa Fluor® 594 azide (A10270) and alkyne (A10275)• Alexa Fluor® 647 azide (A10277) and alkyne (A10278)• Oregon Green® 488 azide (O10180) and alkyne (O10181)• Tetramethylrhodamine (TAMRA) azide (T10182) and alkyne (T10183)• Biotin azide (B10184, Figure 3.1.3) and alkyne (B10185, Figure 3.1.4)

Antibodies to Oregon Green® 488, tetramethylrhodamine and Alexa Fluor® 488 dyes (Section 7.4) and Tyramide Signal Ampli�cation (TSA™) Kits (Section 6.2) are available to provide signal ampli�cation if necessary.11 �e biotin azide and alkyne reagents facilitate western blotting ap-plications and streptavidin enrichment in combination with our streptavidin or CaptAvidin™ agarose 12–14 (S951, C21386; Section 7.6).

Azide- and Alkyne-Modi�ed Nucleosides and Amino AcidsWe o�er the following azide- and alkyne-modi�ed nucleosides and amino acids for

Click-iT® labeling protocols:

• 5-ethynyl uridine (EU, E10345)• 5-ethynyl-2 -́deoxyuridine (EdU; A10044, E10187, E10415; Figure 3.1.1A)• Click-iT® AHA (L-azidohomoalanine for nascent protein synthesis, C10102)• Click-iT® HPG (L-homopropargylglycine for nascent protein synthesis, C10186; Figure

3.1.1B)

�e alkyne-modi�ed nucleosides EdU and EU form the basis of our Click-iT® cell prolif-eration and nascent RNA assays described below. �e individual packagings of these reagents provide the larger quantities required for in vivo labeling applications.6,15 AHA and HPG are methionine surrogates providing nonradioactive alternatives to 35S-methionine for pulse-chase detection of protein synthesis and degradation.16–19

Figure 3.1.4 Biotin alkyne (PEG4 carboxamide-propargyl biotin, B10185).

(CH2)4

S

HN

NH

O

H

H

H

CO

NH(CH2CH2O)4CH2CH2 NHCH2C CHC

O

Figure 3.1.3 Biotin azide (PEG4 carboxamide-6-azidohexa-nyl biotin, B10184).

(CH2)4

S

HN

NH

O

H

H

H

CO

NH(CH2CH2O)4CH2CH2 NH(CH2)6 N NNC

O








Azide- and Alkyne-Modi�ed Monosaccharides, Fatty Acids and Isoprenoids�e Click-iT® metabolic glycoprotein labeling reagents provide biosynthetic precursors for

detecting and characterizing post-translational glycosylation of proteins:14,20–22

• Click-iT® GalNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azido acetyl-galactosamine for labeling O-linked glycoproteins, C33365; Figure 3.1.1C, Figure 3.1.5)

• Click-iT® ManNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetyl-mannosamine for labeling sialic acid–modi�ed glycoproteins, C33366; Figure 3.1.6)

• Click-iT® GlcNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylglucosamine for labeling O-linked N-acetylglucosamine (O-GlcNAc)–modi�ed glycoproteins, C33367; Figure 3.1.7)

• Click-iT® fucose alkyne (tetraacetylfucose alkyne, C10264; Figure 3.1.8)

Cultured cells are simply incubated with the modi�ed sugars for 2–3 days or until cells reach the appropriate density. �e acetyl groups improve cell permeability of the modi�ed sug-ars and are removed by nonspeci�c intracellular esterases (Figure 3.1.8). �e resulting azide- or alkyne-modi�ed sugar is then metabolically incorporated through the permissive nature of the oligosaccharide biosynthesis pathway, yielding functionalized glycoproteins that can be che-moselectively coupled to complementary alkyne- or azide-functionalized �uorophores and bio-tinylation reagents for detection or a�nity capture. We also o�er the the Click-iT® O-GlcNAc Enzymatic Labeling System for in vitro enzyme-mediated N-azidoacetylgalactosamine labeling of O-GlcNAc–modi�ed glycoproteins 26,27 (C33368, Section 9.4) and Click-iT® Protein Analysis Detection Kits (C33370, C33371, C33372; Section 9.4) for detection of azide-functionalized gly-coproteins in 1D or 2D electrophoresis gels or western blots.

Similarly, our azide-functionalized isoprenoids and fatty acids enable detection of post-translational lipidation of proteins by in-gel �uorescence scanning, �uorescence microscopy and �ow cytometry.23–25 We o�er the following azide-modi�ed fatty acids and isoprenoids:

• Click-iT® farnesyl alcohol, azide (C10248, Figure 3.1.9)• Click-iT® geranylgeranyl alcohol, azide (C10249, Figure 3.1.10)• Click-iT® palmitic acid, azide (15-azidopentadecanoic acid, C10265; Figure 3.1.1D)• Click-iT® myristic acid, azide (12-azidododecanoic acid, C10268)

Heterobifunctional ReagentsOur collection of heterobifunctional reagents provide a means for adapting the amine- and

thiol-reactive labeling chemistries described in Chapters 1 and 2 with the azide–alkyne Click-iT® labeling protocols:

• Azide succinimidyl ester (A10280, Figure 3.1.11)• Alkyne succinimidyl ester (A10279, Figure 3.1.12)• Azide iodoacetamide (I10188, Figure 3.1.13)• Alkyne iodoacetamide (I10189, Figure 3.1.14)

Figure 3.1.6 Metabolic incorporation of tetraacetylated azido sugars.

OH3CCO

HN

H2C

H3CCO

O

H3CCO

O

CH2 N3C

O

OCCH3

O

O

OHO

HN

H2C

HO

HO

CH2 N3C

O

OH

OHO

HN

H2C

HO

HO

CH2 N3C

O

O

Tetraacetylated azido sugar(ManNAz)

Cell membrane

Metabolicincorporation

Nonspeci�c esterasescleave acetates, leaving

hydroxyls

Protein

Protein

Figure 3.1.8 Click-iT® fucose alkyne (tetraacetylfucose alkyne, C10264).

OOCCH3

CCH

OCCH3

O

OCCH3

OO

OCCH3

O

Figure 3.17 Click-iT® GlcNAz metabolic glycoprotein label-ing reagent (tetraacetylated N-azidoacetylglucosamine for labeling O-linked N-acetylglucosamine (O-GlcNAc)–modi-�ed glycoproteins; C33367).

OCH3COCH3CO

NH

CH2

OCCH3

O

OCCH2N N N

O

OCCH3

O

O

Figure 3.1.5 Click-iT® GalNAz metabolic glycoprotein label-ing reagent (tetraacetylated N-azidoacetylgalactosamine, C33365).

OCH3CO

NH

CH2

OCCH3

CCH2N

OCCH3CH3CO

O

O

O

O

O

N N







Figure 3.1.11 Azido (PEO)4 propionic acid, succinimidyl ester (3-(azidotetra(ethyleneoxy))propionic acid, succinimi-dyl ester, A10280).

NN N O

O

N

O

O

(CH2CH2O)4CH2CH2 C

Figure 3.1.12 Alkyne, succinimidyl ester (3-propargyloxy-propanoic acid, succinimidyl ester, A10279).

O

O

N

O

O

CCH2OCH2CH2 CCH

�e succinimidyl ester reagents can be used for azide or alkyne functionalization of amine-containing molecules and molecular assemblies including terminally or internally modi�ed oli-gonucleotides and nanoparticles.8,28

Click-iT® Reaction Bu�ersFor added convenience, we o�er Click-iT® Reaction Bu�er Kits for protein or cell samples

labeled with an azide- or alkyne-tagged biomolecule. �e Click-iT® Cell Reaction Bu�er Kit (C10269) includes su�cient reagents to perform 50 reactions based on a 0.5 mL reaction volume for subsequent analyses by �ow cytometry, �uorescence microscopy or high-content screening (HCS). �e Click-iT® Protein Reaction Bu�er Kit (C10276) includes everything required for click coupling to functionalized proteins for subsequent standard protein biochemical analyses (e.g., western blots or mass spectrometry).

Click-iT® Tools for Cell Proliferation Analysis�e Click-iT® EdU cell proliferation assay provides a superior alternative to bromodeoxyuri-

dine (BrdU) or 3H-thymidine incorporation methods for measuring new DNA synthesis.29,30 �e alkynyl nucleoside analog EdU (5-ethynyl-2 -́deoxyuridine; A10044, E10187, E10415) is incorpo-rated into DNA during the synthesis phase (S phase) of the cell cycle and is subsequently detected by copper (I)–catalyzed click coupling to an azide-derivatized �uorophore 6 (Figure 3.1.2). �e small size of the click-coupled �uorophore compared to that of antibodies required for immuno-detection of BrdU (Figure 3.31) enables e�cient penetration of complex samples without the need for harsh cell treatment, simplifying the assay considerably. �e Click-iT® EdU assay protocol is compatible with both adherent cells and cell suspensions. From start to �nish, the EdU detection assay is complete in as little as 90 minutes, as compared with the antibody-based BrdU method, which takes 6–24 hours to complete. In addition, the Click-iT® EdU cell proliferation assay can be multiplexed with surface and intracellular marker detection using Alexa Fluor® dye–labeled secondary antibodies 15,31–33 (Section 7.2) (Figure 3.1.15). Although the majority of applications are in cultured mammalian cells, Click-iT® EdU reagents and methods have also been success-fully applied to a wide range of model organisms including:

• Escherichia coli 34

• Caenorhabditis elegans 32

• Drosophila 35

• Zebra�sh 36

• Mouse 15

• Plants 37,38 (alfalfa, Arabidopsis, grape, maize, rice and tobacco)

Figure 3.1.13 Iodoacetamide azide (I10188).

NH(CH2)6C

O

N N NICH2

Figure 3.1.14 Iodoacetamide alkyne (I10189).

NHCH2CC

O

ICH2 CH

Figure 3.1.15 Multicolor imaging with the Click-iT® EdU Imaging Kits. Muntjac cells were treated with 10 µM EdU for 45 minutes. Cells were then �xed and permeabilized, and EdU that had been incorporated into newly synthesized DNA was detected by the far-red–�uorescent Click-iT® EdU Alexa Fluor® 647 HCS Assay Kit (C10356, C10357). Tubulin was labeled with an anti-tubulin antibody and visualized with an Alexa Fluor® 350 goat anti–mouse IgG antibody (A21049). The Golgi complex was stained with the green-�uorescent Alexa Fluor® 488 conjugate of lectin HPA from Helix pomatia (edible snail) (L11271), and peroxisomes were labeled with an anti-peroxisome antibody and visualized with an orange-�uorescent Alexa Fluor® 555 donkey anti–rabbit IgG antibody (A31572).

Figure 3.1.10 Click-iT® geranylgeranyl alcohol, azide (C10249).

N N NCH2(C CHCH2CH2)3C

CH3 CH3

CHCH2OH

Figure 3.1.9 Click-iT® farnesyl alcohol, azide (C10248).

N N NCH2(C CHCH2CH2)2C

CH3 CH3

CHCH2OH








Figure 3.1.16 EdUTP nucleotide, provided in the Click-iT® TUNEL Imaging Assay Kits.

HN

O

OC CH

N

OCH2POPOPO

HOO O O

O OO

OH3 (CH3CH2)3NH

Figure 3.1.17 5-Ethynyl uridine (EU, E10345).

N

O

OH

HOCH2

HN

O

O

C CH

OH

Click-iT® EdU Flow Cytometry Assay Kits�e Click-iT® EdU Flow Cytometry Assay Kits provide all the reagents needed to perform

50 assays using 0.5 mL reaction bu�er per assay, including the nucleoside analog EdU and all components for �xation, permeabilization and labeling whole blood samples, adherent cells or suspension cells.:30,39

• Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (C35002)• Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit (A10202)• Click-iT® EdU Paci�c Blue™ Flow Cytometry Assay Kit (A10034)

Additionally, two cell-cycle stains compatible with the �uorescence excitation and emission characteristics of the �uorescent azide detection reagents are included.

Click-iT® EdU Imaging Kits�e Click-iT® EdU Imaging Kits contain all of the components needed to label and detect

incorporated EdU on 50 coverslips using 0.5 mL reaction bu�er per test, as well as the blue-�uorescent Hoechst 33342 nuclear stain for identi�cation of cells irrespective of EdU incorpora-tion status:

• Click-iT® EdU Alexa Fluor® 488 Imaging Kit (C10337)• Click-iT® EdU Alexa Fluor® 555 Imaging Kit (C10338)• Click-iT® EdU Alexa Fluor® 594 Imaging Kit (C10339)• Click-iT® EdU Alexa Fluor® 647 Imaging Kit (C10340)

Click-iT® EdU HCS Assay Kits�e Click-iT® EdU HCS Assay Kits contain all of the materials needed to label and detect

incorporated EdU in adherent cells in 96-well microplates and 100 µL reaction bu�er per assay:

• Click-iT® EdU Alexa Fluor® 488 HCS Assay Kit (2-plate size, C10350; 10-plate size, C10351)• Click-iT® EdU Alexa Fluor® 555 HCS Assay Kit (2-plate size, C10352; 10-plate size, C10353)• Click-iT® EdU Alexa Fluor® 594 HCS Assay Kit (2-plate size, C10354; 10-plate size, C10355)• Click-iT® EdU Alexa Fluor® 647 HCS Assay Kit (2-plate size, C10356; 10-plate size, C10357;

Figure 3.1.15)

For cell registration or DNA pro�ling, these kits also include the blue-�uorescent HCS NuclearMask™ Blue stain.

Click-iT® EdU Microplate Assay Kit�e Click-iT® EdU Microplate Assay Kit provides a simple and rapid work�ow with fewer

wash steps resulting in a substantial time-savings advantage over traditional BrdU colorimetric or �uorescent cell proliferation assays. �is assay uses Oregon Green® 488 azide for click cou-pling to synthetically incorporated EdU. �e signal is ampli�ed using immunodetection of the Oregon Green® 488 �uorophore by a rabbit anti–Oregon Green® horseradish peroxidase (HRP) conjugate followed by �uorogenic or chromogenic detection with our Amplex® UltraRed HRP substrate. �e Click-iT® EdU microplate assay has been successfully tested in HeLa, A549, U2OS and A541 cells with a variety of reagents that modulate DNA synthesis, including the DNA syn-thesis inhibitor aphidicolin and the mitotic inhibitor paclitaxel. �e Click-iT® EdU Microplate Assay Kit (C10214) contains su�cient reagents for performing 400 individual assays in a 96-well plate format.

Click-iT® TUNEL Assay�e terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay—based

on the incorporation of modi�ed dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3 -́OH ends of fragmented DNA—is probably the most widely used in situ test for study-ing apoptotic DNA fragmentation. For a sensitive and reliable TUNEL imaging assay, it is vital that the modi�ed nucleotide is an e�cient substrate for TdT. �e minimally modi�ed EdUTP







nucleotide (Figure 3.1.16) used in the Click-iT® TUNEL imaging assay is rapidly incorporated by TdT, allowing samples to be rapidly �xed in order to preserve late-stage apoptotic cells, thereby lessening the possibility of false-negative results due to cell detachment and subsequent loss. �e enzymatically incorporated nucleotide is detected by copper (I)–catalyzed click coupling to an azide-derivatized �uorophore. Compared with assays that use one-step incorporation of dye-modi�ed nucleotides, the fast and sensitive Click-iT® TUNEL imaging assay can detect a higher percentage of apoptotic cells under identical conditions in 2 hours or less.

�e Click-iT® TUNEL Imaging Assay Kits are available with a choice of azide-derivatized Alexa Fluor® dyes, providing �exibility for combination with other apoptosis detection reagents (Section 15.5):

• Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay (C10245)• Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay (C10246)• Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay (C10247)

�e Click-iT® TUNEL assay has been tested in HeLa, A549 and CHO K1 cells with a variety of reagents that induce apoptosis, including staurosporine, and multiplexed with antibody-based de-tection of other apoptosis biomarkers such as cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3 and phosphohistone 2B. It has also proven e�ective for detection of apoptosis induced by siRNA knockdown of the DEC2 transcription factor in human MCF-7 breast cancer cells.40

Click-iT® Tools for Detecting RNA and Protein SynthesisClick-iT® RNA Kits

Click-iT® RNA Imaging and Click-iT® RNA HCS Assay Kits provide everything needed to detect newly synthesized RNA in adherent cells.:

• Click-iT® RNA Alexa Fluor® 488 Imaging Kit (C10329)• Click-iT® RNA Alexa Fluor® 594 Imaging Kit (C10330)• Click-iT® RNA Alexa Fluor® 488 HCS Assay (C10327)• Click-iT® RNA Alexa Fluor® 594 HCS Assay (C10328)

Click-iT® RNA assays are ideal for imaging global RNA synthesis in multiplex analyses us-ing traditional �uorescence microscopy or high-content screening (HCS). �e Click-iT® RNA assays employ the alkyne-modi�ed nucleoside EU (5-ethynyl uridine, E10345; Figure 3.1.17), which is supplied to cells and incorporated into nascent RNA.41 �e small size of the alkyne tag enables e�cient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes. Detection of incorporated EU is accomplished by copper (I)–catalyzed click coupling to an azide-derivatized �uorophore. �e multiplexing capability of the assays makes them ideal for toxicological pro�ling or interrogation of disease models using high-content imaging platforms.

�e Click-iT® RNA HCS Assay Kits (C10329, C10330) contain su�cient reagents to label and detect newly synthesized RNA in whole cells using two 96-well microplates and 50 µL reaction volumes per well. �ese kits also supply the blue-�uorescent HCS NuclearMask™ blue stain as a nuclear counterstain for cell demarcation or for DNA pro�ling. �e Click-iT® RNA Imaging Kits (C10327, C10328) contain su�cient reagents to label and detect newly synthesized RNA in whole cells using 25 coverslips and 500 µL reaction volume per well. �ese kits also supply the blue-�uorescent Hoechst 33342 dye as a nuclear counterstain or for DNA pro�ling.

�e Click-iT® Nascent RNA Capture Kit (C10365) enables RNA synthesized during a time win-dow de�ned by administration of EU to be selectively biotinylated via click coupling of EU to biotin azide. Biotinylated RNA is then captured using streptavidin-functionalized magnetic beads for re-verse transcription and subsequent analysis by DNA sequencing, PCR or microarray hybridization.

Click-iT® Nascent Protein Synthesis Detection KitDetecting newly synthesized protein is key for researchers studying protein biosynthesis, traf-

�cking and degradation. Click-iT® AHA (L-azidohomoalanine, Figure 3.1.18) incorporation pro-vides a fast, sensitive and nonradioactive alternative to the traditional radioactive 35S-methionine

Figure 3.1.18 Click-iT® AHA (L-azidohomoalanine, C10102).

CH

NH2

NCH2CH2 C OH

O

NN

CF3COOH








DATA TABLE 3.1 CLICK CHEMISTRYCat. No. MW Storage Soluble Abs EC Em Solvent NotesA10044 252.23 F,D DMSO, H2O <300 none 1A10266 861.04 F,D,L DMSO 493 76,000 516 pH 7A10267 773.91 F,D,L DMSO 494 76,000 520 pH 7A10270 948.16 F,D,L DMSO 590 105,000 617 pH 7A10275 962.23 F,D,L DMSO 588 100,000 616 pH 7A10277 ~850 F,D,L DMSO 646 270,000 668 MeOHA10278 ~800 F,D,L DMSO 646 250,000 661 pH 7A10279 225.20 F,D DMSO <300 noneA10280 388.38 F,D,L DMSO <300 noneA20012 ~850 F,D,L DMSO 554 151,000 568 pH 7A20013 ~750 F,D,L DMSO 554 150,000 567 pH 7B10184 615.79 F,D,L DMSO <300 noneB10185 528.66 F,D DMSO <300 noneC10102 258.16 F,DD DMSO <300 noneC10186 127.14 F,D DMSO <300 noneC10248 263.38 F,D,LL DMSO <300 noneC10249 331.50 F,D,LL DMSO <300 noneC10264 342.30 F,D DMSO <300 none 2C10265 283.41 F,D,L DMSO <300 noneC10268 241.33 F,D,L DMSO <300 noneC33365 430.37 F,D DMSO <300 none 3C33366 430.37 F,D DMSO <300 none 3C33367 430.37 F,D DMSO <300 none 3E10345 268.23 F,D DMSO <300 noneI10188 310.14 F,D,L DMSO <300 none 4I10189 223.01 F,D,L DMSO <300 none 4O10180 637.68 F,D,L DMSO 494 80,000 521 pH 9 5O10181 449.37 F,D,L DMSO 494 80,000 521 pH 9 5T10182 554.65 F,D,L DMSO 546 95,000 571 MeOH 6T10183 467.52 F,D,L DMSO 543 95,000 572 MeOH 6For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. E10187 and E10415 are an alternative packaging sof A10044 but are otherwise identical.2. C10264 is deacetylated in vivo yielding the 6-ethynyl analog of fucose (MW = 174.15).3. C33365, C33366 and C33367 are deacetylated in vivo yielding N-azidoacetylgalactosamine, N-azidoacetylmannosamine and N-azidoacetylglucosamine (all MW = 262.22), respectively.4. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.5. Absorption and �uorescence of Oregon Green® 488 dyes are pH dependent only in moderately acidic solutions (pH <5).6. Abs and Em for tetramethylrhodamine dyes in pH 8 bu�er are red-shifted approximately 8 nm compared to MeOH, with EC lower by ~10%.

1. Angew Chem Int Ed Engl (2009) 48:6974; 2. Biochemistry (2009) 48:6571; 3. ACS Chem Biol (2006) 1:644; 4. Chem Rev (2009) 109:4207; 5. Chem Rev (2008) 108:2952; 6. Proc Natl Acad Sci U S A (2008) 105:2415; 7. Protein Sci (2004) 13:2693; 8. Angew Chem Int Ed Engl (2009) 48:9879; 9. Bioconjug Chem (2009) 20:728; 10. Methods Enzymol (1990) 186:161; 11. J Histochem Cytochem (2010) 58:207; 12. J Am Chem Soc (2010) 132:2504; 13. Chem Res Toxicol (2008) 21:432; 14. Nat Cell Biol (2008) 10:1224; 15. J Neurosci Methods (2009) 177:122; 16. Yeast (2008) 25:775; 17. Bioorg Med Chem Lett (2008) 18:5995; 18. Proc Natl Acad Sci U S A (2006) 103:9482; 19. Nat Protoc (2007) 2:532; 20. J Virol (2009) 83:13042; 21. Methods Enzymol (2006) 415:230;

22. Proc Natl Acad Sci U S A (2007) 104:2614; 23. J Am Chem Soc (2009) 131:4967; 24. J Am Chem Soc (2007) 129:2744; 25. Proc Natl Acad Sci U S A (2004) 101:12479; 26. J Biol Chem (2009) 284:21327; 27. J Am Chem Soc (2008) 130:11576; 28. Bioconjug Chem (2008) 19:1570; 29. J Immunol Methods (2009) 350:29; 30. Biotechniques (2008) 44:927; 31. J Biol Chem (2010) 285:1544; 32. Genetics (2009) 183:233; 33. Nucleic Acids Res (2009) 37:e31; 34. Methods (2009) 48:8; 35. Science (2009) 325:340; 36. Proc Natl Acad Sci U S A (2009) 106:14896; 37. Plant Methods (2010) 6:5; 38. Proc Natl Acad Sci U S A (2009) 106:11806; 39. Cytometry A (2009) 75:862; 40. Genes Cells (2010) 15:315; 41. Proc Natl Acad Sci U S A (2008) 105:15779; 42. J Neurosci (2009) 29:638.

REFERENCES

technique for the detection of nascent protein.19,42L-azidohomoalanine, an analog of L-methionine, is supplied to cultured cells and is biosyn-thetically incorporated into proteins. �e incorporated amino acid is then detected by copper (I)–catalyzed click coupling to an alkyne-derivatized �uorophore. �is two-step labeling and detection method provides detection sensitivity comparable with that obtained using the radioactive 35S-methionine method and is compatible with down-stream LC-MS/MS and MALDI-MS analysis. Click-iT® AHA is avail-able as a stand-alone reagent (C10102) or in the Click-iT® AHA Alexa

Fluor® 488 Protein Synthesis HCS Assay Kit (C10289), which contains Alexa Fluor® 488 alkyne for detection. Click-iT® AHA has proven to be a successful substitute for methionine in many cell types, including COS-7, 3T3-L1, HeLa, HEK 293 and Jurkat cells. Cellular incorporation of Click-iT® AHA should be carried out in methionine-free media, as methionine is the preferred substrate for methionyl tRNA transferase, and supplemented media (i.e., methionine-free DMEM) should be used in place of HBSS to achieve greater Click-iT® AHA incorporation at lower concentrations.







PRODUCT LIST 3.1 CLICK CHEMISTRYCat. No. Product QuantityA10267 Alexa Fluor® 488 alkyne (Alexa Fluor® 488 5-carboxamido-(propargyl), bis(triethylammonium salt)) *5-isomer* 0.5 mgA20013 Alexa Fluor® 555 alkyne, triethylammonium salt 0.5 mgA10275 Alexa Fluor® 594 alkyne (Alexa Fluor® 594 carboxamido-(5-(and 6-)propargyl), bis(triethylammonium salt)) *mixed isomers* 0.5 mgA10278 Alexa Fluor® 647 alkyne, triethylammonium salt 0.5 mgA10266 Alexa Fluor® 488 azide (Alexa Fluor® 488 5-carboxamido-(6-azidohexanyl), bis(triethylammonium salt)) *5-isomer* 0.5 mgA20012 Alexa Fluor® 555 azide, triethylammonium salt 0.5 mgA10270 Alexa Fluor® 594 azide (Alexa Fluor® 594 carboxamido-(6-azidohexanyl), triethylammonium salt) *mixed isomers* 0.5 mgA10277 Alexa Fluor® 647 azide, triethylammonium salt 0.5 mgA10279 alkyne, succinimidyl ester (3-propargyloxypropanoic acid, succinimidyl ester) 1 mgA10280 azido (PEO)4 propionic acid, succinimidyl ester (3-(azidotetra(ethyleneoxy))propionic acid, succinimidyl ester) 1 mgB10185 biotin alkyne (PEG4 carboxamide-propargyl biotin) 1 mgB10184 biotin azide (PEG4 carboxamide-6-azidohexanyl biotin) 1 mgC10102 Click-iT® AHA (L-azidohomoalanine) *for nascent protein synthesis* 5 mgC10289 Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay *2-plate size* 1 kitC10269 Click-iT® Cell Reaction Bu�er Kit 1 kitC35002 Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit *50 assays* 1 kitA10202 Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit *50 assays* 1 kitA10034 Click-iT® EdU Paci�c Blue™ Flow Cytometry Assay Kit *50 assays* 1 kitC10350 Click-iT® EdU Alexa Fluor® 488 HCS Assay *2-plate size* 1 kitC10351 Click-iT® EdU Alexa Fluor® 488 HCS Assay *10-plate size* 1 kitC10352 Click-iT® EdU Alexa Fluor® 555 HCS Assay *2-plate size* 1 kitC10353 Click-iT® EdU Alexa Fluor® 555 HCS Assay *10-plate size* 1 kitC10354 Click-iT® EdU Alexa Fluor® 594 HCS Assay *2-plate size* 1 kitC10355 Click-iT® EdU Alexa Fluor® 594 HCS Assay *10-plate size* 1 kitC10356 Click-iT® EdU Alexa Fluor® 647 HCS Assay *2-plate size* 1 kitC10357 Click-iT® EdU Alexa Fluor® 647 HCS Assay *10-plate size* 1 kitC10337 Click-iT® EdU Alexa Fluor® 488 Imaging Kit *for 50 coverslips* 1 kitC10338 Click-iT® EdU Alexa Fluor® 555 Imaging Kit *for 50 coverslips* 1 kitC10339 Click-iT® EdU Alexa Fluor® 594 Imaging Kit *for 50 coverslips* 1 kitC10340 Click-iT® EdU Alexa Fluor® 647 Imaging Kit *for 50 coverslips* 1 kitC10214 Click-iT® EdU Microplate Assay *400 assays* 1 kitC10248 Click-iT® farnesyl alcohol, azide *mixed isomers* 1 mgC10264 Click-iT® fucose alkyne (tetraacetylfucose alkyne) 5 mgC33365 Click-iT® GalNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylgalactosamine) *for O-linked glycoproteins* *5.2 mg* eachC10249 Click-iT® geranylgeranyl alcohol, azide *mixed isomers* 1 mgC33367 Click-iT® GlcNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetylglucosamine) *for O-GlcNAc-modi�ed proteins* *5.2 mg* eachC10186 Click-iT® HPG (L-homopropargylglycine) *for nascent protein synthesis* 5 mgC33366 Click-iT® ManNAz metabolic glycoprotein labeling reagent (tetraacetylated N-azidoacetyl-d-mannosamine) *for sialic acid glycoproteins* *5.2 mg* eachC10268 Click-iT® myristic acid, azide (12-azidododecanoic acid) 1 mgC10365 Click-iT® Nascent RNA Capture Kit *for gene expression analysis* 1 kitC10265 Click-iT® palmitic acid, azide (15-azidopentadecanoic acid) 1 mgC10276 Click-iT® Protein Reaction Bu�er Kit 1 kitC10327 Click-iT® RNA Alexa Fluor® 488 HCS Assay *2-plate size* 1 kitC10328 Click-iT® RNA Alexa Fluor® 594 HCS Assay *2-plate size* 1 kitC10329 Click-iT® RNA Alexa Fluor® 488 Imaging Kit *for 25 coverslips* 1 kitC10330 Click-iT® RNA Alexa Fluor® 594 Imaging Kit *for 25 coverslips* 1 kitC10245 Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay *for microscopy and HCS* *50–100 assays* 1 kitC10246 Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay *for microscopy and HCS* *50–100 assays* 1 kitC10247 Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay *for microscopy and HCS* *50–100 assays* 1 kitA10044 EdU (5-ethynyl-2’-deoxyuridine) 50 mgE10187 EdU (5-ethynyl-2’-deoxyuridine) 500 mgE10415 EdU (5-ethynyl-2’-deoxyuridine) 5 gE10345 5-ethynyl uridine (EU) 5 mgI10189 iodoacetamide alkyne 1 mgI10188 iodoacetamide azide 1 mgO10181 Oregon Green® 488 alkyne *6-isomer* 0.5 mgO10180 Oregon Green® 488 azide (Oregon Green® 6-carboxamido-(6-azidohexanyl), triethylammonium salt) *6-isomer* 0.5 mgT10183 tetramethylrhodamine (TAMRA) alkyne (5-carboxytetramethylrhodamine, propargylamide) *5-isomer* 0.5 mgT10182 tetramethylrhodamine (TAMRA) azide (tetramethylrhodamine 5-carboxamido-(6-azidohexanyl)) *5-isomer* 0.5 mg







Section 3.2 Reagents for Modifying Alcohols

Figure 3.2.1 Sodium periodate oxidation of an N-terminal serine residue to an aldehyde, with the release of formalde-hyde. The aldehyde thus formed from the protein can be subsequently modi�ed with a variety of hydrazine, hydroxylamine or amine derivatives.

Terminal serine

NH2 C

CH2

OH

C R

O

NaIO4HC C

O

O

+ HC H

O

H

Alcohols in Proteins: Serine, Threonine and Tyrosine Residues

Although alcohols (including phenols such as tyrosine and the hydroxyl groups in serine, threonine, sterols and carbohydrates) are abundant in biomolecules, their chemical reactivity in aqueous solution is extremely low. Few reagents are selective for alcohols in aqueous solution, especially in the presence of more reactive nucleophiles such as thiols and amines. It is therefore di�cult to selectively modify serine, threonine and tyrosine residues in proteins except when they exhibit unusual reactivity, such as by residing at an enzyme’s active site.

Serine and Threonine ResiduesNonacylated N-terminal serine and threonine residues in peptides and proteins can be oxi-

dized with periodate to yield aldehydes 1,2 (Figure 3.2.1) that can be subsequently modi�ed with a variety of hydrazine, hydroxylamine or amine derivatives (Section 3.3, Table 3.2). In addition, peptides containing serine, threonine or tyrosine residues separated from a histidine residue by a single amino acid can be selectively acylated by the succinimidyl ester or sulfosuccinimidyl ester of biotin-X 3 (B1582, B6353; Section 4.2; Figure 3.2.2). �is property may also permit selec-tive modi�cation of these sequences (Ser-x-His, �r-x-His and Tyr-x-His, where “x” refers to any amino acid) in peptides and proteins with �uorescent succinimidyl esters (Chapter 1). O-acylation versus N-acylation can be detected by treatment with hydroxylamine, which cleaves esters but usually not amides.4

Figure 3.2.2 Nucleophilic attack of serine on the carbonyl group (C=O) of biotin-X, SSE (B6353) results in the stable O-acylated derivative. In addition to histidine-x-serine, this stable intermediate can be formed in the presence of linear se-quences of histidine-x-tyrosine and histidine-x-threonine, where “x” refers to any amino acid.

H

R2H

C

H

N

O

C

X

C

H

N

O

C

H

N N

CH2CN

CH2

O

C

O

(CH2)5NH

R1H

C

O

(CH2)5NH

CH2)4

R2H

C

H

N

O

C

X

C

H

N

O

C

H

N N

CH2CN

CH2

O

H

H

R1

NH

N

H

H

H

O

NH

N

H

H

H

O

(CH2)4NH(CH2)5 C

O

NH(CH2)5 C

O

O

O

O

SO3

)

+ _

N

Figure 3.2.3 4-Chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD chloride; 4-chloro-7-nitrobenzofurazan, C20260).

3.2 Reagents for Modifying Alcohols







Tyrosine ResiduesModi�cation of tyrosine residues is sometimes a side reaction when proteins are reacted with

sulfonyl chlorides, iodoacetamides or other reactive dyes described in Chapter 1 and Chapter 2. For example, NBD chloride (C20260, Section 1.8, Figure 3.2.3) reacts with an active-site tyro-sine in Escherichia coli F1-ATPase, causing strong inhibition.5

Tyrosine residues in some proteins can be selectively modi�ed by initial nitration of the ortho position of its phenol using tetranitromethane, and then reduction of the o-nitrotyrosine with sodium dithionite (Na2S2O4) to form an o-aminotyrosine (Figure 3.2.4). Although much less reactive than aliphatic amines, the aromatic amine of o-aminotyrosine can react with most amine-reactive reagents (Chapter 1) between pH 5 and pH 7.6 To obtain selective derivatiza-tion in complex samples, it is of course critical to block all aliphatic primary amines, typically by acetylation with acetic anhydride, prior to reduction of nitrotyrosine. Nitration of tyrosine residues by nitric oxide occurs naturally in cells through peroxynitrite radical intermediates (Section 18.3, Table 18.1), yielding derivatives that can be reduced to o-aminotyrosine and subse-quently detected using amine-reactive reagents.6,7 Tyrosine residues of peptides and proteins can be selectively coupled to aniline derivatives in the presence of formaldehyde.8 Chemoselective derivatization of tyrosine has also been accomplished using a �uorescent diazodicarboxamide reagent prepared from carboxy-X-rhodamine (ROX) succinimidyl ester 9 (C1309, Section 1.6).

Another method for modifying tyrosine groups in peptides is to convert the phenol group in tyrosine residues to a salicylaldehyde derivative, and then to react the salicylaldehyde with 1,2-di-amino-4,5-dimethoxybenzene (D1463, Section 3.3) to form a �uorescent benzimidazole.10–12

�e tyramide signal ampli�cation (TSA™) technology (Section 6.2), which was developed by NEN (now a part of PerkinElmer Corporation) and licensed for in-cell and in-tissue applications, permits signi�cant ampli�cation of the detectability of targets by a horseradish peroxidase–me-diated scheme. In the TSA™ method, the labeled tyramide becomes covalently linked to tyrosine residues in or near the target. We have introduced an extensive selection of TSA™ Kits that utilize an Alexa Fluor® tyramide, Oregon Green® 488 tyramide or biotin-XX tyramide as the ampli�ca-tion reagents (Section 6.2).

Alcohols in CarbohydratesAs with derivatization of alcohols in proteins, it is di�cult to selectively modify most carbo-

hydrates in aqueous solution because of their low reactivity and the competing hydrolysis of the reactive reagents. However, several reagents are available for derivatizing reducing sugars (which contain a low equilibrium concentration of the reactive aldehyde function), as well as for modify-ing aldehydes and ketones obtained by periodate oxidation of various carbohydrates. To pursue this labeling approach, see Section 3.3 for a description of aldehyde- and ketone-reactive reagents.

DichlorotriazinesDichlorotriazines readily modify amines in proteins, and they are among the few reactive

groups that are reported to react directly with polysaccharides and other alcohols in aque-ous solution, provided that the pH is >9 and that other nucleophiles are absent. We o�er the 5-isomer of �uorescein dichlorotriazine (5-DTAF, D16), with absorption/emission maxima of ~492/516 nm, as well as Texas Red® C2-dichlorotriazine (T30200, Figure 3.2.5), with absorption/emission maxima of ~588/601 nm. 5-DTAF has been used to label a wide range of hydroxylated biopolymers including collagen,13 cellulose,14 cyclodextrins 15 and soluble beta-glucan,16 as well as functionalized carbon nanotubes.17

N-Methylisatoic AnhydrideIn the absence of other reactive functional groups, N-methylisatoic anhydride (M25) will

convert ribonucleotides and certain other carbohydrates 18,19 to �uorescent esters with excita-tion/emission maxima of ~350/446 nm in mildly basic aqueous solution.20–24 �e compactness and moderate environmental sensitivity of this �uorophore, which is a synthetic precursor to blue-�uorescent N-methylanthraniloyl (MANT) amides and esters,23 may be advantageous for preparing site-selective probes. Low molecular weight alcohols are better derivatized by this reagent in aprotic organic solvents 25–27 (Figure 3.2.6).

Figure 3.2.4 Reaction scheme for the conversion of tyro-sine to o-aminotyrosine. Tyrosine undergoes nitration by re-action with tetranitromethane, followed by reduction with sodium dithionite, to yield an o-aminotyrosine.

C

NO2

N

NO2O2N

O2NH

CH2C

O

OH

OH

NO2

NH

CH2C

O

Na2S2O4

OH

NH2

NH

CH2C

O

+CH

CH

CH

R1

R1

R1

R2

R2

R2

Figure 3.2.5 Texas Red® C2-dichlorotriazine (T30200).

N O N

2

4

SO3

SO2NHCH2CH2NH

NN

N

Cl

Cl

Figure 3.2.6 Reaction of N-methylisatoic anhydride (M25) with an alcohol to produce a blue-�uorescent (~350/446 nm) N-methylanthraniloyl (MANT) ester.

O

N

O O

CH3

R OHC

OO R

NHCH3








Dansyl Aminophenylboronic Acidm-Dansylaminophenylboronic acid (D2281, Figure 3.2.7) reacts with vicinal diols (hy-

droxyl groups on adjacent carbon atoms) and certain amino alcohols 28 to form cyclic com-plexes 29 (Figure 3.2.8) that have a �uorescence intensity and peak emission dependent on the environment of the dansyl �uorophore. �is interesting reagent binds reversibly to cell-wall carbohydrates,30 as well as to glycosylated (but not deglycosylated) human serum albumin.31 Dansylaminophenylboronic acid is also used as an HPLC derivatization reagent for vicinal di-ols 32 and as a detection reagent for glycolipids analyzed by thin-layer chromatography.33

Alcohol Modi�cation in Organic SolventsTwo functional groups—acyl azides and acyl nitriles—react directly with aliphatic amines

to yield the same products as do the corresponding succinimidyl esters. When reacted in organic solvents, however, these reagents can also form derivatives of alcohols and phenols, making them extremely useful for sensitive analysis of alcohols by HPLC or capillary electrophoresis.

Isocyanates Prepared from Acyl AzidesAlcohols are much easier to modify in anhydrous organic solvents than in aqueous solu-

tion. Perhaps the most e�ective reagents are isocyanates, which are much more reactive with alcohols (and amines) than are isothiocyanates but are not su�ciently stable to permit their sale. Fortunately, isocyanates can o�en be prepared by Curtius rearrangement of acyl azides (Figure 3.2.9). When an acyl azide and alcohol are heated together in an organic solvent such as toluene, dioxane or DMF at 80°C, the acyl azide will rearrange to form an isocyanate that then reacts with the alcohol to form a stable urethane. As little as 50 femtograms of the urethane con-jugates prepared from coumarin derivatives 7-methoxycoumarin-3-carbonyl azide (M1445) and 7-diethylaminocoumarin-3-carbonyl azide (D1446) can be detected using an HPLC �uorescence detector.34,35 Alcohol conjugates (urethanes) prepared from the single-isomer carbonyl azides of �uorescein diacetate (F6218) and tetramethylrhodamine (T6219) may provide even higher sen-sitivity, particularly with instruments that employ the argon-ion laser. Following rearrangement and alcohol conjugation, the acetates of the �uorescein derivative can be removed by hydrolysis at pH 9–10. �e diacetate of �uorescein-5-carbonyl azide has been used to synthesize a �uorogenic substrate for the anandamide transmembrane carrier.36 Tetramethylrhodamine-5-carbonyl azide has been successfully conjugated to the hydrophobic poly(ε-caprolactone) (PCL) block of a diblock copolymer micelle in order to follow its cellular internalization 37,38 and has also been used to pre-pare ribo�avin conjugates for the same purpose.39,40

Acyl Nitriles9-Anthroylnitrile (A1440) reacts with alcohols, such as steroids and acylglycerols, in organic

solvents to yield carboxylate esters that are useful for HPLC.41,42 To optimize solid-phase organic synthesis, 9-anthroylnitrile has been used to quantitate the absolute amount of resin-bound hy-droxyl groups directly on solid support.43 In addition, 9-anthroylnitrile has been reported to be useful for the selective labeling of certain serine and threonine residues in myosin.44–48 �e lipo-philicity of 9-anthroylnitrile may make it useful for modifying hydroxyl groups of proteins and hydroxylated fatty acids that are buried within cell membranes.

Figure 3.2.7 m-Dansylaminophenylboronic acid (D2281).

Figure 3.2.8 Reaction of m-dansylaminophenylboronic acid (D2281) with a vicinal diol to form a reversible �uores-cent cyclic complex.

N(CH3)2

SO2NH B(OH2)

C C

OH OH

N(CH3)2

SO2NH

O

BO

+ H H

H

H

R1

R1

R2

R2

Figure 3.2.9 Derivatization of an alcohol using the diacetate of �uorescein-5-carbonyl azide (F6218). This process consists of three steps: 1) rearrangement of the acyl azide to an isocyanate, 2) reaction of the isocyanate with an alcohol to form a urethane and 3) deprotection of the non�uorescent urethane derivative using hydroxylamine.

1 32







DATA TABLE 3.2 REAGENTS FOR MODIFYING ALCOHOLSCat. No. MW Storage Soluble Abs EC Em Solvent NotesA1440 231.25 F,D,L DMF, MeCN 361 7500 470 MeOH 1D16 495.28 F,D,L pH >6, DMF 492 83,000 516 pH 9 2, 3D1446 286.29 F,D,L DMF, MeCN 436 57,000 478 MeOHD2281 370.23 D,L DMF, DMSO 337 4600 517 MeOH 4F6218 485.41 FF,D DMF, MeCN <300 noneM25 177.16 D DMF, DMSO 316 3500 386 MeOH 5M1445 245.19 FF,D,L DMF, MeCN 360 25,000 415 MeOHT6219 455.47 FF,D,L DMF, MeCN 545 90,000 578 MeOHT30200 796.74 F,D,L DMF, DMSO 583 87,000 604 MeOHFor de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. The absorption spectrum of A1440 has subsidiary peaks at 380 nm and 344 nm. Emission spectrum is unstructured. Ester derivatives formed by reaction with alcohols have essentially similar spectra.

2. Unstable in water. Use immediately.3. Absorption and �uorescence of �uorescein derivatives are pH dependent. Extinction coe�cients and �uorescence quantum yields decrease markedly at pH <7.4. Fluorescence of D2281 when bound to proteins is typically blue shifted (Em ~490 nm).5. The amide reaction product of M25 with butylamine has Abs = 353 nm (EC = 5900 cm–1M–1), Em = 426 nm in MeOH. Ester reaction products with alcohols have Abs = 350 nm

(EC = 5700 cm–1M–1), Em = 446 nm in water (pH 8).

PRODUCT LIST 3.2 REAGENTS FOR MODIFYING ALCOHOLSCat. No. Product Quantity

A1440 9-anthroylnitrile 25 mgD2281 m-dansylaminophenylboronic acid 100 mgD16 5-(4,6-dichlorotriazinyl)amino�uorescein (5-DTAF) *single isomer* 100 mgD1446 7-diethylaminocoumarin-3-carbonyl azide 25 mgF6218 �uorescein-5-carbonyl azide, diacetate 10 mgM1445 7-methoxycoumarin-3-carbonyl azide 25 mgM25 N-methylisatoic anhydride *high purity* 1 gT6219 tetramethylrhodamine-5-carbonyl azide 5 mgT30200 Texas Red® C2-dichlorotriazine 5 mg

REFERENCES1. Bioconjug Chem (2003) 14:205; 2. Bioconjug Chem (1992) 3:138; 3. Anal Biochem (2010) 398:123; 4. J Biol Chem (1992) 267:5060; 5. J Biol Chem (2004) 279:31505; 6. J Proteome Res (2007) 6:2257; 7. Anal Biochem (1998) 259:127; 8. Bioconjug Chem (2008) 19:153; 9. J Am Chem Soc (2010) 132:1523; 10. J Chromatogr (1988) 430:271; 11. J Chromatogr (1986) 356:171; 12. J Chromatogr (1985) 344:267; 13. J Biomech (2009) 42:2279; 14. Biomacromolecules (2003) 4:481; 15. Biomacromolecules (2009) 10:505; 16. J Immunol (2006) 177:1661; 17. Langmuir (2009) 25:7573; 18. Electrophoresis (2007) 28:1221; 19. Anal Biochem (2000) 284:167; 20. Biochemistry (1991) 30:422; 21. Biochemistry (1990) 29:3309; 22. Biochim Biophys Acta (1983) 742:496; 23. J Biol Chem (1982) 257:13354; 24. Arch Biochem Biophys (1973) 155:70;

25. Anal Biochem (1996) 234:31; 26. Synthesis (1982) 39:266; 27. J Org Chem (1959) 24:1214; 28. Anal Bioanal Chem (2006) 386:506; 29. Med Res Rev (2010) 30:171; 30. Biochem Biophys Res Commun (1980) 96:157; 31. Clin Chim Acta (1985) 149:13; 32. Anal Chim Acta (1990) 228:101; 33. J Lipid Res (1995) 36:1848; 34. Chem Pharm Bull (1985) 33:1164; 35. J Lipid Res (1997) 38:429; 36. J Pharmacol Exp �er (2000) 293:289; 37. Science (2003) 300:615; 38. Bioconjug Chem (2002) 13:1259; 39. Mol Pharm (2004) 1:257; 40. J Pharmacol Exp �er (2003) 306:681; 41. Anal Chim Acta (1983) 147:397; 42. J Chromatogr (1983) 276:289; 43. Anal Chem (1999) 71:4564; 44. Biochemistry (1996) 35:16061; 45. Biochemistry (1994) 33:6867; 46. J Biol Chem (1990) 265:18791; 47. J Biol Chem (1990) 265:18786; 48. J Biol Chem (1989) 264:18188.







Section 3.3 Reagents for Modifying Aldehydes and Ketones

Aldehydes and ketones are present in a number of low molecular weight molecules such as drugs, steroid hormones, reducing sugars and metabolic intermediates (e.g., pyruvate and α-ketoglutarate). Except for polysaccharides containing free reducing sugars, however, biopolymers generally lack aldehyde and ketone groups. Even those aldehydes and ketones that are found in the open-ring form of simple carbohydrates are usually in equilibrium with the closed-ring form of the sugar.

�e infrequent occurrence of aldehydes and ketones in biomole-cules has stimulated the development of techniques to selectively intro-duce these functional groups, thus providing unique sites for chemical modi�cation and greatly extending the applications of the probes found in this section. Fluorescent modi�cation of aldehyde or carboxylic acid groups in carbohydrates is also frequently utilized for their analysis by HPLC, capillary electrophoresis 1 and other methods.

Introducing Aldehydes and Ketones into BiomoleculesPeriodate Oxidation

�e most common method for introducing aldehydes and ketones into polysaccharides and glycoproteins (including antibodies) is by periodate-mediated oxidation of vicinal diols. �ese introduced alde-hydes and ketones can then be modi�ed with �uorescent or biotinylated hydrazine, hydroxylamine or amine derivatives to label the polysac-charide or glycoprotein. For example, some of the hydrazine derivatives described in this section have been used to detect periodate-oxidized glycoproteins in gels.2 �e Pro-Q® Emerald 300 and Pro-Q® Emerald 488 Glycoprotein Gel and Blot Stain Kits (P21855, P21857, M33307;

3.3 Reagents for Modifying Aldehydes and Ketones

Figure 3.3.1 Sodium periodate oxidation of an N-terminal serine residue to an aldehyde, with the release of formaldehyde. The aldehyde thus formed from the protein can be subse-quently modi�ed with a variety of hydrazine, hydroxylamine or amine derivatives.

Terminal serine

NH2 C

CH2

OH

C R

O

NaIO4HC C

O

O

+ HC H

O

H

Figure 3.3.2 Oxidation of the terminal galactose residue of a glycoprotein, glycolipid or polysaccharide resultvs in the generation of an aldehyde, which can react with hydrazines, hydroxylamines or primary amine–containing compounds.

H2O2O2

OHO

OH

HO

O

CH

O R

ROHO

CH2OH

OH

HO

O R

= glycolipid, polysaccharide or glycoproteinR

Galactose Oxidase

Section 9.4) are based on periodate oxidation of glycoproteins and sub-sequent labeling with a Pro-Q® Emerald dye.3

Periodate oxidation of the 3 -́terminal ribose provides one of the few methods of selectively modifying RNA; periodate-oxidized ribo-nucleotides can subsequently be converted to �uorescent nucleic acid probes by reaction with �uorescent hydrazines, hydroxylamines and amines.4–6 Alkenes from unsaturated fatty acids and ceramides can also be converted to glycols by osmium tetroxide and then oxidized by peri-odate to aldehydes, and periodate will oxidize certain β-aminoethanol derivatives such as the hydroxylysine residues in collagen, as well as me-thionine (to its sulfoxide) and certain thiols (usually to disul�des). �ese other reactions, however, usually occur at a slower rate than oxidation of vicinal diols.

In addition to vicinal diols, N-terminal serine and threonine resi-dues of peptides and proteins can be selectively oxidized by periodate to aldehyde groups 7–9 (Figure 3.3.1). Moreover, because antibodies are glycosylated at sites distant from the antigen-binding region, modi�ca-tion of periodate-oxidized antibodies by hydrazines 10 and hydroxyl-amines usually does not inactivate the antibody, as sometimes occurs with amine-reactive labeling.

Galactose Oxidase–Mediated Oxidation and Other Methods

Galactose oxidase oxidizes terminal galactose residues to aldehydes, particularly in glycoproteins.11–15 �e introduction of galactose residues can be especially advantageous for structural studies because it pro-vides a means of selectively labeling speci�c sites on biomolecules. For example, 2-keto-galactose has been speci�cally inserted into the Fc gly-cans of therapeutic antibodies, including Herceptin and Avastin, en-abling site-speci�c labeling with Alexa Fluor® 488 hydroxylamine 16,17 (A30629). Galactose oxidase–modi�ed lipopolysaccharides (LPS) have been modi�ed with Alexa Fluor® 488 hydrazide (A10436) to probe for LPS-binding sites on cells.18 Because galactose oxidase–mediated oxida-tion liberates a molecule of hydrogen peroxide for each molecule of al-dehyde that is formed (Figure 3.3.2), horseradish peroxidase–catalyzed oxidation of the Amplex® Red reagent to red-�uorescent resoru�n by hydrogen peroxide provides a ready means by which the number of al-dehyde residues introduced into a biomolecule, including on a cell sur-face, can be quantitated. �e Amplex® Red Galactose/Galactose Oxidase Assay Kit (A22179, Section 10.5) provides the reagents and a general protocol for this assay of introduced aldehyde residues. Other meth-ods for aldehyde and ketone introduction include selective N-terminal transamination in the presence of pyridoxal-5 -́phosphate,19 ligation of a ketone analog of biotin to proteins with a biotin acceptor peptide (BAP) fusion tag by biotin ligase (BirA) 20 and co-translational modi-�cation of recombinantly tagged proteins by formylglycine-generating enzyme 21 (FGE).

Figure 3.3.3 N-(t-BOC)-aminooxyacetic acid, tetra�uorophenyl ester (B30300).

(CH3)3CO C

O

NHOCH2 C O

O

FF

F F







Coupling Hydrazines and Amines to Amine-Containing Biomolecules without Introducing Aldehydes and Ketones

Common tissue �xatives such as formaldehyde and glutaraldehyde can be used to couple hydrazine and amine derivatives to proteins and other amine-containing polymers. For ex-ample, lucifer yellow CH (L453) can be conjugated to surrounding biomolecules by common aldehyde-based �xatives in order to preserve the dye’s staining pattern during subsequent tissue manipulations.22

Introducing a Hydroxylamine into a Biomolecule�e tetra�uorophenyl (TFP) ester of N-(t-BOC)-aminooxyacetic acid (B30300, Figure 3.3.3)

is an amine-reactive protected hydroxylamine that is useful for synthesizing new aldehyde- and ketone-reactive probes in an organic solvent. Following coupling to aliphatic amines, the t-BOC group can be quantitatively removed with tri�uoroacetic acid. �e resultant hydroxylamine probe can then spontaneously react with aldehydes, the reducing ends of saccharides and oligo-saccharides, and abasic sites in oligonucleotides to form stable adducts.

Hydrazines and HydroxylaminesReactivity of Hydrazine and Hydroxylamine Derivatives

Although certain aromatic amines such as 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, A350), 2-aminoacridone (A6289) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS, A6257; Figure 3.3.4) have been extensively utilized to modify reducing sugars for analysis and sequencing, the most reactive reagents for forming stable conjugates of aldehydes and ketones are usually hydrazine derivatives, including hydrazides, semicarbazides and carbohydrazides (Figure 3.3.5), as well as hydroxylamine derivatives. Hydrazine derivatives react with ketones to yield relatively stable hydrazones (Figure 3.3.6), and with aldehydes to yield hydrazones that are somewhat less stable, though they may be formed faster. Hydroxylamine derivatives (aminooxy compounds) react with aldehydes and ketones to yield oximes. Oximes are superior to hydra-zones with respect to hydrolytic stability.23 Both hydrazones and oximes can be reduced with sodium borohydride (NaBH4) to further increase the stability of the linkage. Rates and yields of aldehyde reactions with hydrazine and hydroxylamine derivatives are substantially enhanced by aniline catalysis.24 �is chemistry is su�ciently mild and e�cient to be applicable for labeling periodate-oxidized sialylated glycoproteins on the surface of live cells.25

Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with Visible Light

We o�er a large number of �uorescent hydrazine and hydroxylamine derivatives for reac-tion with aldehydes or ketones (Table 3.2). Because they are more photostable than the �uorescein derivatives, the Alexa Fluor®, BODIPY® and Texas Red® hydrazides should be among the most sensitive reagents for detecting aldehydes and ketones in laser-excited chromatographic methods.26 However, with the exception of the Alexa Fluor® 555 and Alexa Fluor® 647 hydrazides and the Alexa Fluor® 647 hydroxylamine, the Alexa Fluor® reagents are mixed isomers and may resolve into mul-tiple peaks when analyzed with high-resolution separation techniques. Fluorescent hydrazides and hydroxylamines are extensively used for labeling glycans via derivatization of aldehydes generated a�er periodate oxidation or via coupling to the reducing terminus.27,28 Alexa Fluor® 488 hydrox-ylamine (A30629, Figure 3.3.7) is particularly useful for detecting aldehyde groups at abasic DNA lesions,29,30 similar to the biotinylated hydroxylamine ARP described later in this section.

Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with UV LightDansyl hydrazine (D100) has been by far the most widely used UV light–excitable hydrazine

probe for derivatizing aldehydes and ketones for chromatographic analysis and mass spectrom-etry.31,32 A unique application that has been reported for dansyl hydrazine, but that is likely a gen-eral reaction of hydrazine derivatives, is the detection of N-acetylated or N-formylated proteins through transfer of the acyl group to the �uorescent hydrazide.33,34 Although dansyl hydrazine has been widely used as a UV light–excitable derivatization reagent, our 7-diethylaminocoumarin

Figure 3.3.4 8-Aminopyrene-1,3,6-trisulfonic acid, triso-dium salt (APTS, A6257).

Figure 3.3.5 Structures of A) a hydrazide, B) a semicarba-zide and C) a carbohydrazide.

Hydrazide

Semicarbazide

Carbohydrazide

A

B

C

R NH CNHNH2

O

R NHNH2

NHNH CNHNH2

O

R

C

O

Figure 3.3.6 Modifying aldehydes and ketones with hydra-zine derivatives.

CR1

R2O CR1

R2NNHR3

R3NHNH2

Figure 3.3.7 Alexa Fluor® 488 C5-aminooxyacetamide, bis (triethylammonium) salt (Alexa Fluor® 488 hydroxylamine, A30629).

O

NH(CH2)5NH

H2N O NH2

C

O

O

SO3SO3

2 (CH3CH2)3NHC

O

NH2OCH2 C

6

5








Figure 3.3.8 Absorption and �uorescence emission spectra of lucifer yellow CH in water.

Table 3.2 Molecular Probes® hydrazine, hydroxylamine and amine derivatives.

Derivative Hydrazines * Hydroxylamines * Cadaverines * Other Amines *

Acridone A6289 †

Alexa Fluor® 350 A10439 A30627 A30674

Alexa Fluor® 405 A30675

Alexa Fluor® 488 A10436 A30629 A30676

Alexa Fluor® 555 A20501MP A30677

Alexa Fluor® 568 A10437 A30680

Alexa Fluor® 594 A10438 A30678

Alexa Fluor® 633 A30634

Alexa Fluor® 647 A20502 A30632 A30679

Bimane B30633

Biotin and DSB-X™ biotin ‡ B1603, B2600, D20653 A10550 § A1594, B1596 A1593, B1592, N6356

t-BOC B30300 ** M6248

BODIPY® FL D2371 D2390

BODIPY® TR D6251

Cascade Blue® C687 C621

Coumarin D355 A191 †

Dansyl D100 D113 D112

Dapoxyl® D10460

2,3-Diaminonaphthalene D7918 †

Dimethoxybenzene D1463 †

Dinitrophenyl D1552 *

Fluorescein C356, F121 A10466 A1351, A1353, A1363

Lucifer yellow L453 A1340 A1339

Naphthalene A91, A350 †

NBD M20490

Oregon Green® 488 O10465

Pyrene P101 A6257 †

QSY® 7 Q10464

Rhodamine A1318 L2424

Texas Red® T6256 T2425

* Hydrazine, hydroxylamine and aromatic amine derivatives are discussed in Section 3.3, and aliphatic amine derivatives are discussed in Section 3.4, except for D1552, which appears in Section 12.3. † Aromatic amines used extensively for modifying aldehydes and ketones. ‡ Biotin and desthiobiotin (DSB-X™ biotin) derivatives are listed in Section 4.2. § ARP is the abbreviation for N-(aminooxyacetyl)-N’-(D-biotinoyl)hydrazine. ** B30300 requires t-BOC deprotection by tri�uoroacetic acid to generate free hydroxylamine. More information on reactive dyes is available at www.invitrogen.com/handbook/labelingchemistry.

and pyrene hydrazides (D355, P101) have much higher absorptivity and �uorescence, which should make their conjugates more detectable than those of dansyl hydrazine.

Polar Fluorescent Hydrazides and HydroxylaminesLucifer yellow CH (L453) is most commonly used as an aldehyde-�xable neuronal tracer

with visible absorption and emission (Figure 3.3.8). �is membrane-impermeant hydrazide also reacts with periodate-oxidized cell-surface glycoproteins,35,36 oxidized ribonucleotides 37 and gangliosides.38 Cascade Blue® hydrazide (C687) exhibits high absorptivity (EC >28,000 cm–1M–1), �uorescence quantum yield (0.54) and water solubility 39 (~1%). Like Cascade Blue® hydrazide, Alexa Fluor® 350 hydrazide (A10439) and Alexa Fluor® 350 hydroxylamine (A30627) also have high water solubility and bright blue �uorescence. �ese sulfonated pyrene and coumarin deriva-tives have applications similar to those of lucifer yellow CH, including as aldehyde-�xable polar tracers; 40 see Section 14.3 for a more complete discussion of this application.

Cell membrane–impermeant aldehyde- and ketone-reactive reagents are also important probes for assessing the topology of peptide and protein exposure on the surface of live cells. Periodate- or galactose oxidase–mediated oxidation of cell-surface glycoproteins and polysaccharides can be used to selectively introduce aldehyde residues on the cell surface, and these aldehydes can then be reacted with a membrane-impermeant hydrazide. �e high polarity of our Alexa Fluor® hydrazides (A10436, A10437, A10438, A10439, A20501MP, A20502, A30634), Alexa Fluor® hydroxylamines







Figure 3.3.9 Reaction scheme illustrating the principle of ketone and aldehyde detection by NBD methylhydrazine (M20490).

N

O

N

NO2

N NH2CH3

N

O

N

NO2

N NCH3 CR1

R2C

R1

R2O

R1, R2 = H, alkyl, aryl

(A30627, A30629, A30632), lucifer yellow CH (L453) and Cascade Blue® hydrazide (C687) make them the preferred labeling reagents.

NBD MethylhydrazineNBD methylhydrazine (N-methyl-4-hydrazino-7-

nitrobenzofurazan, M20490) has been used to monitor aldehydes and ketones in tobacco smoke 41 and automobile exhaust 42 and also to mea-sure nitrite in water 43 (Section 21.2). NBD methylhydrazine reacts with carbonyl compounds in acidic media, forming the corresponding hy-drazones (Figure 3.3.9). Following separation by HPLC, the hydrazones can be detected either by spectrophotometry (using wavelengths cor-responding to the absorption maxima of the relevant hydrazone) or by �uorescence spectroscopy using excitation/emission at ~470/560 nm.

Biotin Hydrazides and Biotin HydroxylamineIn addition to the �uorescent hydrazine and hydroxylamine de-

rivatives, we o�er several non�uorescent biotin and DSB-X™ biotin hydrazides (B1603, B2600, D20653; Section 4.2) and the biotin hydrox-ylamine derivative ARP (A10550, Section 4.2), each of which can be detected using �uorescent dye– or enzyme-labeled avidin or strep-tavidin (Section 7.6, Table 7.9). DSB-X™ biotin hydrazide, which has moderate a�nity for avidin and streptavidin that is rapidly reversed by low concentrations of free biotin, can be used to produce a DSB-X™ biotin–labeled molecule that reversibly binds avidin or streptavidin af-�nity matrices (Section 7.6).

We recommend the biotin hydroxylamine derivative ARP (alde-hyde-reactive probe, A10550; Figure 3.3.10) as our most e�cient re-agent for incorporating biotins into aldehyde- or ketone-containing cell surfaces. ARP has been used extensively to modify the exposed aldehyde group at abasic lesions in DNA 44,45 (Figure 3.3.11). A quick and sensitive microplate assay for abasic sites can be performed using ARP.46 In addition, ARP is membrane permeant, permitting detection

Figure 3.3.10 N-(aminooxyacetyl)-N’-(D-biotinoyl) hydrazine, tri�uoroacetic acid salt (ARP, A10550).

Figure 3.3.11 Aldehyde-reactive probe (ARP) used to detect DNA damage. The biotin hy-droxylamine ARP (A10550) reacts with aldehyde groups formed when reactive oxygen spe-cies depurinate DNA. This reaction forms a covalent bond linking the DNA to biotin. The bio-tin can then be detected using �uorophore- or enzyme-linked streptavidin.

5′

3′

NH

NHNH

SH

(CH2)4C

O

H

H

NH

C

O

NH2OCH2

O

+

ARP

5′

3′

5′

3′

N

N

N

N

NH2

CH2O

O

O

P

O

O O−

CH2

CHOH

O

O

P

O

O O−

O

O

O

C

NH

H

H

O

C

(CH2)4

HS

NHNH

NH

NOCH2

O−

O

O

P

O

O

OHCH

CH2

Reactive Oxygen Species (ROS)or Radiation

of abasic sites in live cells.47,48 Once the aldehyde groups in abasic sites are modi�ed by ARP and the cells are �xed and permeabilized, the re-sulting biotinylated DNA can be detected with �uorescent dye–, Qdot® nanocrystal– or enzyme-conjugated streptavidin conjugates (Section 7.6, Table 7.9). Likewise, ARP can be used to detect and capture 4-hy-droxynonenal (HNE)–modi�ed proteins.49 ARP has also been used to immobilize IgG antibodies on streptavidin-coated monolayer surfaces with their binding sites oriented toward the solution phase.50 An alter-native to ARP for detection of protein carbonyls is dinitrophenylhydra-zine derivatization followed by immunolabeling with our Alexa Fluor® 488 dye–labeled anti-dinitrophenyl antibody 51 (A11097, Section 7.4).








Aliphatic and Aromatic AminesPrimary aliphatic and aromatic amines (Table 3.2) can be cou-

pled reversibly to aldehydes and ketones to form hydrolytically un-stable Schi� bases 11 (Figure 3.3.12). �e reversibility of this modi�-cation makes reagents that contain amines less desirable unless the Schi� base is reduced by sodium borohydride 52,53 or sodium cyano-borohydride.54 Chemical reduction also retains the amine’s original charge. Sequencing of carbohydrate polymers using �uorescent de-rivatives has usually relied on derivatization of the reducing end of the polymer with a �uorescent amine.55 Certain aromatic amines have been extensively utilized for coupling to aldehydes, ketones, monosac-charides and the reducing end of carbohydrate polymers:

• 2-Aminoacridone (A6289) forms conjugates that can be separated by HPLC 56,57 or, as their borate complexes, by polyacrylamide gel electrophoresis,55,58,59 capillary electrophoresis 60–62 and micellar electrokinetic capillary chromatography 63,64 (MECC). Starting with as little as 25 µg of a glycoprotein, researchers have e�-ciently released and puri�ed the carbohydrates, and then de-rivatized them with 2-aminoacridone for subsequent structural analysis.65 2-Aminoacridone derivatives of oligosaccharides have

been directly analyzed by MALDI-TOF mass spectrometry.66 2-Aminoacridone is also used to prepare �uorogenic substrates for proteases.67

• 7-Amino-4-methylcoumarin (A191), which is a common base of protease substrates (Section 10.4), can be used for the reductive derivatization of oligosaccharides.68,69

• 8-Aminopyrene-1,3,6-trisulfonic acid (APTS, A6257) has been extensively used to derivatize carbohydrates prior to separation by gel or capillary electrophoresis.70,71 Among the amines we o�er, APTS is the aromatic amine that has the most favorable combina-tion of strong absorbance, high quantum yield and ionic charge.

• ANTS (A350) has a high ionic charge, permitting electrophoretic separation of its products with complex oligosaccharides.72

�e aromatic diamine 1,2-diamino-4,5-dimethoxybenzene (DDB, D1463), which forms heterocyclic compounds with certain aldehydes and ketones, has been used to selectively detect aromatic aldehydes in the presence of aliphatic aldehydes, including carbohydrates.73 DBB has proven to be a useful reagent for HPLC analysis of the cytotoxic metabolic by-product methylglyoxal in blood samples from diabetic patients.74

Alternatively, aldehydes and ketones can be transformed into pri-mary aliphatic amines by reductive amination with ammonia, eth-ylenediamine or other non�uorescent diamines.75 �is chemistry is particularly useful because the products can then be coupled with any of the amine-reactive reagents described in Chapter 1 such as the succinimidyl esters of TAMRA dye 76 (C1171, C6121, C6122; Section 1.6). Derivatization by succinimidyl esters has been extensively uti-lized for tagging oligosaccharides that are to be separated by capillary zone electrophoresis with laser-induced �uorescence detection.76–79Figure 3.3.12 Modifying aldehydes and ketones with amine derivatives.

CR1

R2NR3

CR1

R2O

NaCNBH3R3NH2 R1 CH

R2NHR3

1. J Biomed Mater Res A (2007) 83:1176; 2. Anal Biochem (1987) 161:245; 3. Electrophoresis (2003) 24:588; 4. Nat Struct Mol Biol (2008) 15:494; 5. Proc Natl Acad Sci U S A (2004) 101:7902; 6. Bioconjug Chem (1994) 5:436; 7. Bioconjug Chem (2003) 14:205; 8. Bioconjug Chem (1992) 3:262; 9. Bioconjug Chem (1994) 5:636; 10. J Immunol (2009) 183:1551; 11. Methods Enzymol (1994) 247:30; 12. Anal Biochem (1988) 170:271; 13. Methods Enzymol (1987) 138:429; 14. Biochem Biophys Res Commun (1980) 92:1215; 15. J Supramol Struct (1977) 6:291; 16. Bioconjug Chem (2009) 20:1228; 17. Bioconjug Chem (2009) 20:1383; 18. Cytometry (2000) 41:316; 19. ACS Chem Biol (2007) 2:247; 20. Mol �er (2008) 16:1467; 21. Proc Natl Acad Sci U S A (2009) 106:3000; 22. Nature (1981) 292:17; 23. Angew Chem Int Ed Engl (2008) 47:7523; 24. Bioconjug Chem (2008) 19:2543; 25. Nat Methods (2009) 6:207; 26. Electrophoresis (2007) 29:475; 27. BMC Urol (2005) 5:4; 28. J Am Chem Soc (2009) 131:10360; 29. Anal Bioanal Chem (2007) 387:1883; 30. Angew Chem Int Ed Engl (2007) 46:561; 31. Anal Chem (2010) 82:2893; 32. J Chromatogr A (2002) 979:409; 33. J Cell Biol (1988) 106:1607; 34. Anal Biochem (1969) 29:186; 35. Biochemistry (1985) 24:322; 36. Biochem Biophys Res Commun (1983) 112:872; 37. Biochemistry (1988) 27:6039; 38. J Cell Biol (1985) 100:721; 39. Anal Biochem (1991) 198:119; 40. Physiol Res (1997) 46:407; 41. Fresenius J Anal

Chem (2000) 366:396; 42. Anal Chem (1999) 71:1893; 43. Anal Chem (1999) 71:3003; 44. Photochem Photobiol (2002) 76:123; 45. Anal Chem (2001) 73:2229; 46. Methods (2000) 22:164; 47. Proc Natl Acad Sci U S A (2000) 97:686; 48. J Biol Chem (2000) 275:6741; 49. Anal Chem (2006) 78:6847; 50. Anal Biochem (2003) 312:113; 51. Anal Bioanal Chem (2008) 391:2591; 52. Biochemistry (1987) 26:2162; 53. Biochim Biophys Acta (1980) 597:285; 54. Biochim Biophys Acta (1981) 670:181; 55. Anal Biochem (1994) 222:270; 56. Anal Chem (1998) 70:2530; 57. Anal Chem (1997) 69:4985; 58. Anal Biochem (1994) 216:243; 59. Anal Biochem (1991) 196:238; 60. Anal Biochem (1996) 240:68; 61. Electrophoresis (1996) 17:406; 62. Anal Biochem (1995) 230:115; 63. Anal Chem (1996) 68:4424; 64. J Chem Soc Chem Commun (1994) 14:1691; 65. Anal Biochem (1998) 262:197; 66. Rapid Commun Mass Spectrom (1997) 11:1635; 67. Anal Biochem (1988) 171:393; 68. Biophys J (1996) 71:2040; 69. Anal Biochem (1983) 128:41; 70. Nat Protoc (2006) 1:397; 71. Electrophoresis (2005) 26:2034; 72. Anal Biochem (2000) 283:136; 73. J Chromatogr B Biomed Appl (1994) 659:85; 74. Diabetes Metab (2006) 32:176; 75. Proc Natl Acad Sci U S A (1991) 88:2302; 76. J Chromatogr B Biomed Appl (1994) 657:307; 77. Anal Biochem (1995) 227:368; 78. Carbohydr Res (1996) 296:203; 79. J Biomol Screen (1999) 4:239.

REFERENCES







DATA TABLE 3.3 REAGENTS FOR MODIFYING ALDEHYDES AND KETONESCat. No. MW Storage Soluble Abs EC Em Solvent NotesA191 175.19 L DMF, DMSO 351 18,000 430 MeOHA350 427.33 L H2O 353 7200 520 H2OA6257 523.39 D,L H2O 424 19,000 505 pH 7A6289 246.70 D,L DMF, DMSO 425 5200 531 MeOH 1A10436 570.48 D,L H2O 493 71,000 517 pH 7A10437 730.74 D,L H2O 576 86,000 599 pH 7 2A10438 758.79 D,L H2O 588 97,000 613 pH 7 2A10439 349.29 L H2O, DMSO 345 13,000 445 pH 7A20501MP ~1150 D,L H2O 554 150,000 567 pH 7A20502 ~1200 D,L H2O 649 250,000 666 pH 7A30627 584.52 F,D,L H2O, DMSO 353 20,000 437 MeOH 3A30629 895.07 F,D,L H2O, DMSO 494 77,000 518 pH 7 3, 4, 5A30632 ~1220 F,D,L H2O, DMSO 651 250,000 672 MeOH 3A30634 ~950 D,L H2O, DMSO 624 110,000 643 pH 7B30300 339.24 F,D DMSO <300 noneC356 493.49 L pH >7, DMF 492 78,000 516 pH 8 6C687 596.44 L H2O 399 30,000 421 H2O 7, 8D100 265.33 L EtOH 336 4400 534 MeOHD355 275.31 D,L MeCN, DMF 420 46,000 468 MeOHD1463 241.12 D,L EtOH 298 3100 359 MeOHD2371 306.12 F,D,L MeOH, MeCN 503 71,000 510 MeOH 9D7918 158.20 L DMSO, MeOH 340 5100 377 MeOH 10F121 421.43 D,L pH >7, DMF 492 85,000 516 pH 9 6L453 457.24 L H2O 428 12,000 536 H2O 11, 12M20490 209.16 F,L MeCN 487 24,000 none MeOH 13P101 302.38 D,L MeCN, DMF 341 43,000 376 MeOH 14T6256 620.74 F,L DMF 582 109,000 602 MeOHFor de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. Spectra of this compound are in methanol containing a trace of KOH.2. Maximum solubility in water is ~8% for A10437 and A10438.3. Aqueous stock solutions should be used within 24 hours; long-term storage is NOT recommended.4. The �uorescence lifetime (τ) of the Alexa Fluor® 488 dye in pH 7.4 bu�er at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.5. Abs and Em of the Alexa Fluor® 488 dye are red-shifted by as much as 16 nm and 25 nm respectively on microarrays relative to aqueous solution values. The magnitude of the spectral shift

depends on the array substrate material. (Biotechniques (2005) 38:127)6. Absorption and �uorescence of �uorescein derivatives are pH dependent. Extinction coe�cients and �uorescence quantum yields decrease markedly at pH <7.7. The Alexa Fluor® 405 and Cascade Blue® dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.8. Maximum solubility in water is ~1% for C687, ~1% for C3221 and ~8% for C3239.9. The absorption and �uorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent.10. Fluorescence of D7918 is weak. Reaction with α-ketoaldehydes yields �uorescent benzoquinoxaline derivatives (Abs = 365 nm, Em = 540 nm in H2O at pH 8). (J Chromatogr B Biomed Sci Appl

(1999) 729:237)11. The �uorescence quantum yield of lucifer yellow CH in H2O is 0.21. (J Am Chem Soc (1981) 103:7615)12. Maximum solubility in water is ~8% for L453, ~6% for L682 and ~1% for L1177.13. NBD methylhydrazine reacts with aldehydes and ketones in the presence of strong acid, yielding weakly �uorescent hydrazone products. (Anal Chem (1999) 71:1893) Abs = 493 nm,

Em = 552 nm in MeOH for reaction product with acetone.14. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below

300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.








PRODUCT LIST 3.3 REAGENTS FOR MODIFYING ALDEHYDES AND KETONESCat. No. Product QuantityA30627 Alexa Fluor® 350 C5-aminooxyacetamide, tri�uoroacetate salt (Alexa Fluor® 350 hydroxylamine) 1 mgA30629 Alexa Fluor® 488 C5-aminooxyacetamide, bis(triethylammonium) salt (Alexa Fluor® 488 hydroxylamine) 1 mgA30632 Alexa Fluor® 647 C5-aminooxyacetamide, bis(triethylammonium) salt (Alexa Fluor® 647 hydroxylamine) 1 mgA10439 Alexa Fluor® 350 hydrazide, sodium salt 5 mgA10436 Alexa Fluor® 488 hydrazide, sodium salt 1 mgA20501MP Alexa Fluor® 555 hydrazide, tris(triethylammonium) salt 1 mgA10437 Alexa Fluor® 568 hydrazide, sodium salt 1 mgA10438 Alexa Fluor® 594 hydrazide, sodium salt 1 mgA30634 Alexa Fluor® 633 hydrazide, bis(triethylammonium) salt 1 mgA20502 Alexa Fluor® 647 hydrazide, tris(triethylammonium) salt 1 mgA6289 2-aminoacridone, hydrochloride 25 mgA191 7-amino-4-methylcoumarin *reference standard* 100 mgA350 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) 1 gA6257 8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt (APTS) 10 mgB30300 N-(t-BOC)-aminooxyacetic acid, tetra�uorophenyl ester 25 mgC356 5-(((2-(carbohydrazino)methyl)thio)acetyl)amino�uorescein 25 mgC687 Cascade Blue® hydrazide, trisodium salt 10 mgD1463 1,2-diamino-4,5-dimethoxybenzene, dihydrochloride (DDB) 100 mgD7918 2,3-diaminonaphthalene 100 mgD355 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH) 25 mgD2371 4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY® FL hydrazide) 5 mgD100 5-dimethylaminonaphthalene-1-sulfonyl hydrazine (dansyl hydrazine) 100 mgF121 �uorescein-5-thiosemicarbazide 100 mgL453 lucifer yellow CH, lithium salt 25 mgM20490 N-methyl-4-hydrazino-7-nitrobenzofurazan (NBD methylhydrazine) 25 mgP101 1-pyrenebutanoic acid, hydrazide 100 mgT6256 Texas Red® hydrazide *>90% single isomer* 5 mg






Section 3.4 Derivatization Reagents for Carboxylic Acids and Carboxamides

Figure 3.4.2 Stabilization of an unstable O-acylisourea intermediate by N-hydroxysuccinimide in a carbodiimide-mediated (EDAC, E2247) modi�cation of a carboxylic acid with a primary amine.

NH3+

C

O

NH

HON

O

O

+(CH3)2NH(CH3)2 N C N CH2CH3+

COH

O

(CH3)2NH(CH3)2 N C N CH2CH3

OC

O

+

ON

O

O

C

O

NH3+

C

O

NH

R1R1R1

R1R1

R2

R2R2

R2

Figure 3.4.1 Conversion of a carboxylic acid group into an aliphatic amine. The activated carboxylic acid is derivatized with a half-protected aliphatic diamine (mono-N-(t-BOC)-propylenedi-amine, M6248), usually in an organic solvent, followed by removal of the t-BOC–protecting group with tri�uoroacetic acid.

+ RC OH

O ED C CF3COOHH2N(CH2)3NH CR

O

NH(CH2)3NH2C

O

(CH3)3CO NH(CH2)3NHC

O

(CH3)3CO CR

OA

Carboxylic acids can be converted to esters, amides, acyl hy-drazides or hydroxamic acids, all of which are discussed in this sec-tion. Alternatively, the half-protected tert-butyloxycarbonyl (t-BOC) propylenediamine derivative (M6248) is useful for converting organic solvent–soluble carboxylic acids into aliphatic amines. Following cou-pling of the half-protected aliphatic diamine to an activated carboxylic acid, the t-BOC group can be quantitatively removed with tri�uoroace-tic acid (Figure 3.4.1). �e resultant aliphatic amine can then be modi-�ed with any of the amine-reactive reagents described in Chapter 1 or coupled to solid-phase matrices for a�nity chromatography.

Coupling Hydrazines, Hydroxylamines and Amines to Carboxylic AcidsModi�cation in Aqueous Solutions

�e carboxylic acids of water-soluble biopolymers such as proteins can be coupled to hydrazines, hydroxylamines and amines (Table 3.2) in aqueous solution using water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, E2247). Including N-hydroxysulfosuccinimide (H2249) in the reaction mixture has been shown to improve the coupling e�ciency of EDAC-mediated protein–carboxylic acid conjugations 1,2 (Figure 3.4.2). To reduce intra- and inter-protein coupling to lysine residues,3 which is a common side reaction, carbodiimide-mediated coupling should be performed in a concentrated protein solution at a low pH, using a large excess of the

nucleophile. EDAC-mediated coupling has been used to functionalize Qdot® 605 ITK™ carboxyl quantum dots (Q21301MP, Section 6.6) with the GABA receptor agonist muscimol.4 EDAC has been shown to be impermeable to membranes of live cells, which permits its use to dis-tinguish between cytoplasmic and lumenal sites of reaction.5

Fluoresceinyl glycine amide (5-(aminoacetamido)�uorescein, A1363) and various hydrazines and hydroxylamines may be the best probes for this application because they are more likely to remain reac-tive at a lower pH than are aliphatic amines such as the cadaverines.6 Fluoresceinyl glycine amide has been coupled to the carboxylic acid of a cyclosporin derivative by EDAC.7

ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, A350; Section 3.3) has a high ionic charge, which permits electrophoretic separation of its products with complex oligosaccharides.8 Several of the �uores-cent hydrazine and hydroxylamine derivatives described in Section 3.3 should have similar utility for carbodiimide-mediated derivatization of carboxylic acids.

Modi�cation in Organic SolventsPeptide synthesis research has led to the development of numer-

ous methods for coupling carboxylic acids to amines in organic solu-tion. One such method involves the conversion of carboxylic acids to succinimidyl esters or mixed anhydrides. Dicyclohexylcarbodiimide and diisopropylcarbodiimide are widely used to promote amide forma-tion in organic solution. Another recommended derivatization meth-od for coupling organic solvent–soluble carboxylic acids, including

3.4 Derivatization Reagents for Carboxylic Acids and Carboxamides








Figure 3.4.3 Absorption and �uorescence emission spectra of Dapoxyl® (2-aminoethyl)sulfonamide in methanol.

Figure 3.4.4 Transglutaminase-mediated labeling of a protein using dansyl cadaverine (D113).

N(CH3)2

SO2NH(CH2)5NH2

+ + NH3

N(CH3)2

SO2NH(CH2)5NH C

O

CH2CH2 PROTE I N

H2N C

O

CH2CH2 PROTE INtransglutaminase

peptides, to aliphatic amines without racemization is the combination of 2,2 -́dipyridyldisul�de and triphenylphosphine.9,10 Unlike �uores-cent aliphatic amines, �uorescent aromatic amines such as those de-rived from 7-amino-4-methylcoumarin (A191) and 2-aminoacridone (A6289, Section 3.3) exhibit a shi� in their absorption and emission (if any) to much shorter wavelengths upon forming carboxamides. �is property makes these aromatic amines preferred reagents for preparing peptidase substrates (Section 10.4). Aromatic amines can generally be coupled to acid halides and anhydrides, with organic solvents usually required for e�cient reaction.

Hydrazine, Hydroxylamine and Aliphatic Amine Derivatives

We provide a wide selection of carboxylic acid–reactive reagents (Table 3.2), including several di�erent Dapoxyl®, Alexa Fluor®, BODIPY®, �uorescein, Oregon Green®, rhodamine, Texas Red® and QSY® hydra-zine derivatives, hydroxylamine derivatives and amine derivatives, all of which are particularly useful for synthesizing drug analogs and as probes for �uorescence polarization immunoassays 11–13 (Fluorescence Polarization (FP)—Note 1.4). �ese probes all require a coupling agent such as a carbodiimide to react with carboxylic acids; they do not spon-taneously react with carboxylic acids in solution. �ey do, however, re-act spontaneously with the common amine-reactive functional groups described in Section 1.1, including succinimidyl esters and isothiocya-nates. Some of the more important probes and their applications include:

• Alexa Fluor® hydrazides (A10436, A10437, A10438, A10439, A30634, A20501MP, A20502; Section 3.3), Alexa Fluor® hydroxylamines (A30627, A30629, A30632; Section 3.3) and Alexa Fluor® cadaver-ines (A30674, A30675, A30676, A30677, A30678, A30679, A30680), our brightest and most photostable carboxylic acid–reactive probes

Figure 3.4.5 QSY® 7 amine, hydrochloride (Q10464).

• BODIPY® aliphatic amines (D2390, D6251), for preparing pH-in-sensitive probes, such as BODIPY® FL etoposide,14 from carboxylic acid derivatives

• Isomeric aminomethyl�uoresceins (A1351, A1353), which are read-ily coupled to activated carboxylic acids 15

• Dapoxyl® (2-aminoethyl)sulfonamide (D10460) for preparing con-jugates with strong UV absorption and a Stokes shi� of ~200 nm (Figure 3.4.3)

• Dansyl ethylenediamine (D112), dansyl cadaverine (D113), Dapoxyl® (2-aminoethyl)sulfonamide (D10460) and Lissamine rhodamine B ethylenediamine (L2424), for carboxylic acid de-rivatization 16 and glutamine transamidation reactions 17 (Figure 3.4.4)

• Bimane amine (B30633), a small blue-�uorescent dye for carboxylic acid derivatization

• EDANS (A91), for preparing radioactive IAEDANS,13,18 energy transfer–quenched substrates for endopeptidases 19 (Section 10.4) and an ATP substrate analog for DNA-dependent RNA polymerase 20

• QSY® 7 amine (Q10464, Figure 3.4.5), which is an essentially non-�uorescent dye with strong visible absorption for preparing highly e�cient quenchers for bioassays based on �uorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2)

• Hydrazine (Section 3.3) and amine derivatives of lucifer yellow (A1339), Alexa Fluor® 405 (A30675) and Cascade Blue® (C621) dyes, which are precursors of highly �uorescent, water-soluble probes

• Hydrazine and amine derivatives of biotin and desthiobiotin (Section 4.2), which are versatile intermediates for synthesizing biotin- and desthiobiotin-containing probes 21,22







Figure 3.4.3 Absorption and �uorescence emission spectra of Dapoxyl® (2-aminoethyl)sulfonamide in methanol.

Figure 3.4.4 Transglutaminase-mediated labeling of a protein using dansyl cadaverine (D113).

N(CH3)2

SO2NH(CH2)5NH2

+ + NH3

N(CH3)2

SO2NH(CH2)5NH C

O

CH2CH2 PROTE I N

H2N C

O

CH2CH2 PROTE INtransglutaminase

peptides, to aliphatic amines without racemization is the combination of 2,2 -́dipyridyldisul�de and triphenylphosphine.9,10 Unlike �uores-cent aliphatic amines, �uorescent aromatic amines such as those de-rived from 7-amino-4-methylcoumarin (A191) and 2-aminoacridone (A6289, Section 3.3) exhibit a shi� in their absorption and emission (if any) to much shorter wavelengths upon forming carboxamides. �is property makes these aromatic amines preferred reagents for preparing peptidase substrates (Section 10.4). Aromatic amines can generally be coupled to acid halides and anhydrides, with organic solvents usually required for e�cient reaction.

Hydrazine, Hydroxylamine and Aliphatic Amine Derivatives

We provide a wide selection of carboxylic acid–reactive reagents (Table 3.2), including several di�erent Dapoxyl®, Alexa Fluor®, BODIPY®, �uorescein, Oregon Green®, rhodamine, Texas Red® and QSY® hydra-zine derivatives, hydroxylamine derivatives and amine derivatives, all of which are particularly useful for synthesizing drug analogs and as probes for �uorescence polarization immunoassays 11–13 (Fluorescence Polarization (FP)—Note 1.4). �ese probes all require a coupling agent such as a carbodiimide to react with carboxylic acids; they do not spon-taneously react with carboxylic acids in solution. �ey do, however, re-act spontaneously with the common amine-reactive functional groups described in Section 1.1, including succinimidyl esters and isothiocya-nates. Some of the more important probes and their applications include:

• Alexa Fluor® hydrazides (A10436, A10437, A10438, A10439, A30634, A20501MP, A20502; Section 3.3), Alexa Fluor® hydroxylamines (A30627, A30629, A30632; Section 3.3) and Alexa Fluor® cadaver-ines (A30674, A30675, A30676, A30677, A30678, A30679, A30680), our brightest and most photostable carboxylic acid–reactive probes

Figure 3.4.5 QSY® 7 amine, hydrochloride (Q10464).

• BODIPY® aliphatic amines (D2390, D6251), for preparing pH-in-sensitive probes, such as BODIPY® FL etoposide,14 from carboxylic acid derivatives

• Isomeric aminomethyl�uoresceins (A1351, A1353), which are read-ily coupled to activated carboxylic acids 15

• Dapoxyl® (2-aminoethyl)sulfonamide (D10460) for preparing con-jugates with strong UV absorption and a Stokes shi� of ~200 nm (Figure 3.4.3)

• Dansyl ethylenediamine (D112), dansyl cadaverine (D113), Dapoxyl® (2-aminoethyl)sulfonamide (D10460) and Lissamine rhodamine B ethylenediamine (L2424), for carboxylic acid de-rivatization 16 and glutamine transamidation reactions 17 (Figure 3.4.4)

• Bimane amine (B30633), a small blue-�uorescent dye for carboxylic acid derivatization

• EDANS (A91), for preparing radioactive IAEDANS,13,18 energy transfer–quenched substrates for endopeptidases 19 (Section 10.4) and an ATP substrate analog for DNA-dependent RNA polymerase 20

• QSY® 7 amine (Q10464, Figure 3.4.5), which is an essentially non-�uorescent dye with strong visible absorption for preparing highly e�cient quenchers for bioassays based on �uorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2)

• Hydrazine (Section 3.3) and amine derivatives of lucifer yellow (A1339), Alexa Fluor® 405 (A30675) and Cascade Blue® (C621) dyes, which are precursors of highly �uorescent, water-soluble probes

• Hydrazine and amine derivatives of biotin and desthiobiotin (Section 4.2), which are versatile intermediates for synthesizing biotin- and desthiobiotin-containing probes 21,22





Figure 3.4.6 5-(Bromomethyl)�uorescein (B1355).

OHO O

C OH

O

CH2Br

Enzyme-Catalyzed TransamidationA transglutaminase-catalyzed transamidation reaction of glutamine residues in some pro-

teins and peptides enables their selective modi�cation by amine-containing probes 23,24 (Figure 3.4.4). �is unique method for selective protein modi�cation requires formation of a complex consisting of the glutamine residue, the aliphatic amine probe and the enzyme. It has been found that a short aliphatic spacer in the amine probe enhances the reaction. �e cadaverine (–NH(CH2)5NH–) spacer is usually optimal. Although dansyl cadaverine (D113) has been prob-ably the most widely used reagent,25–27 Alexa Fluor® cadaverines 24 (A30674, A30675, A30676, A30677, A30678, A30679, A30680), Oregon Green® 488 cadaverine (O10465), �uorescein ca-daverine 28–33 (A10466), tetramethylrhodamine cadaverine 34 (A1318), Texas Red® cadaverine (T2425) and BODIPY® TR cadaverine (D6251) are among the most �uorescent transglutaminase substrates available. �e intrinsic transglutaminase activity in sea urchin eggs has been used to covalently incorporate dansyl cadaverine during embryonic development.35 Two biotin cadaver-ines (A1594, B1596; Section 4.2) are also available for transglutaminase-mediated reactions.36–38 Amine-terminated peptides and �uorescent and biotin hydrazides, including Cascade Blue® hy-drazide, have been successfully incorporated into protein fragments by transamidation during enzyme-catalyzed proteolysis.39

Transamidation of cell-surface glutamine residues by the combination of a transgluta-minase enzyme and a �uorescent or biotinylated aliphatic amine can form stable amides.27 Impermeability of the enzyme restricts this reaction to a limited number of proteins on the cell surface. �is technique was used to selectively label erythrocyte band 3 protein with dan-syl cadaverine (D113) and proteins of the extracellular matrix with �uorescein cadaverine 31,32 (A10466). Following protease treatment, the dansylated peptides were isolated using an anti-dansyl a�nity column.40

Esteri�cation of Carboxylic Acids with Fluorescent Diazoalkanes

Biologically important molecules, especially the nonchromophoric fatty acids, bile acids and prostaglandins, are typically esteri�ed by carboxylic acid–reactive reagents in organic sol-vents. Esteri�cation of carboxylic acids in aqueous solution is usually not possible, and esters tend to be unstable in water. Fluorescent derivatization reagents for biomedical chromatography have been extensively discussed in reviews.41,42

HPLC derivatization reagents for carboxylic acids include two �uorescent analogs of the common esteri�cation reagent diazomethane. Diazoalkanes react without the addition of cata-lysts and may be useful for direct carboxylic acid modi�cation of proteins and synthetic poly-mers. Fluorescent diazoalkanes also react with phosphates 43 and potentially with lipid-associat-ed carboxylic acids in membrane-bound proteins or with free fatty acids.

�e �uorescent diazomethyl derivative 9-anthryldiazomethane (ADAM, A1400) has been commonly used to derivatize biomolecules. Unfortunately, ADAM is not very stable and may decompose during storage. 1-Pyrenyldiazomethane 44–47 (PDAM, P1405) is recommended as a replacement for ADAM because it has much better chemical stability. Moreover, the detection limit for PDAM conjugates is reported to be about 20–30 femtomoles, which is �ve times better than reported for detection of ADAM conjugates.45 In addition, fatty acids derivatized with these reagents have been used to measure phospholipase A2 activity 48 (Section 17.4). It has been re-ported that photolysis of pyrenemethyl esters liberates the free carboxylic acid,47 making PDAM a potential protecting group for carboxylic acids.

Fluorescent Alkyl Halides�e low nucleophilicity of carboxylic acids requires that they be converted to anions (typi-

cally cesium or quaternary ammonium are used as counterions) before they can be esteri�ed with alkyl halides in organic solvents. Conjugates of 6-bromoacetyl-2-dimethylaminonaphthalene (badan, B6057) have a high Stokes shi�, as well as spectral properties that are very sensitive

Figure 3.4.8 2-(2,3-Naphthalimino)ethyl tri�uoromethane-sulfonate (N2461).

Figure 3.4.7 BODIPY® 493/503 methyl bromide (8-bromomethyl-4,4-di�uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene, B2103).

NB

N

FF CH3H3C

H3CCH2Br CH3








to their environment. 5-(Bromomethyl)�uorescein (B1355, Figure 3.4.6) and BODIPY® 493/503 methyl bromide (B2103, Figure 3.4.7) have the strongest absorptivity and �uorescence of the currently avail-able carboxylic acid–derivatization reagents.49 An analytical method for estimating the degree of EDAC crosslinking of collagen has been developed based on derivatization of residual carboxyl groups by 5-bromomethylfluorescein.50

All of the alkyl halides in this section also react with thiol groups, including those in proteins.51,52 Although more commonly used as thi-ol-reactive reagents, the monobromobimanes (M1378, M20381; Section 2.3) have been reported to react with carboxylic acids in organic sol-vents.53 �e coumarin iodoacetamide DCIA (D404, Section 2.3) has also been used to derivatize carboxylic acids; 54 other iodoacetamides described in Chapter 2 will probably react similarly.

Figure 3.4.9 4-Sulfo-2,3,5,6-tetra�uorophenol (STP, S10490) can be used to prepare water-soluble activated esters from various carboxylic acids.

F F

F F

SO3-

HO + SO3-

FF

FF

RCOH

O

RCO

O

Fluorescent Tri�uoromethanesulfonate2-(2,3-Naphthalimino)ethyl tri�uoromethanesulfonate (N2461,

Figure 3.4.8) reacts rapidly with the anions of carboxylic acids in aceto-nitrile to give adducts that are reported to be detectable by absorption at 259 nm down to 100 femtomoles and by �uorescence at 394 nm down to 4 femtomoles.55 �is naphthalimide sulfonate ester will likely react with other nucleophiles too, including thiols, amines, phenols (e.g., tyrosine) and probably histidine. 2-(2,3-Naphthalimino)ethyl tri�uoromethane-sulfonate has been used for the sensitive reverse-phase HPLC detection of eicosanoids in brain tissue.56

4-Sulfo-2,3,5,6-Tetra�uorophenol (STP) and N-Hydroxysulfosuccinimide (NHSS)

4-Sulfo-2,3,5,6-tetra�uorophenol (STP, S10490) and N-hydroxysulfosuccinimide (NHSS, H2249) can be used to prepare water-soluble activated esters from various carboxylic acids (Figure 3.4.9). Coupling typically involves a carbodiimide such as EDAC (E2247) and is performed in an organic solvent. We have found that the resulting STP esters are much easier to purify and more stable than activated esters prepared from N-hydroxysulfosuccinimide.57 NHSS esters of biotin and other derivatives considerably increase the aqueous solubility of the reagents.58 We o�er a variety of amine-reactive STP esters, which are discussed in Chapter 1.

REFERENCES1. Bioconjug Chem (2008) 19:1880; 2. Anal Biochem (1986) 156:220; 3. Biophys J (2000) 78:1449; 4. J Am Chem Soc (2006) 128:15701; 5. J Biol Chem (2000) 275:977; 6. Methods Enzymol (1987) 138:429; 7. Bioconjug Chem (1992) 3:32; 8. Anal Biochem (2000) 283:136; 9. J Chromatogr (1993) 645:75; 10. Tetrahedron Lett (1970) 22:1901; 11. Anal Biochem (1987) 162:89; 12. Clin Chem (1985) 31:1193; 13. Biochemistry (1973) 12:4154; 14. Biochem Pharmacol (1997) 53:715; 15. Bioorg Med Chem Lett (2002) 12:1283; 16. Biochim Biophys Acta (1991) 1085:223; 17. Anal Biochem (1971) 44:221; 18. J Labelled Compounds Radiopharmaceut (1983) 20:1265; 19. J Biol Chem (2004) 279:20775; 20. Arch Biochem Biophys (1986) 246:564; 21. J Histochem Cytochem (1990) 38:377; 22. Ann NY Acad Sci (1984) 463:214; 23. J Am Chem Soc (2006) 128:4542; 24. Protein Sci (2006) 15:640; 25. Anal Biochem (1992) 201:270; 26. FEBS Lett (1991) 278:51; 27. Biochemistry (1978) 17:2163; 28. Toxicol in Vitro (1999) 13:773; 29. J Biol Chem (1999) 274:430; 30. Biochem J (1998) 331:105; 31. Exp Cell

Res (1998) 239:119; 32. Histochem J (1997) 29:593; 33. Biochemistry (1988) 27:3483; 34. Biochemistry (1988) 27:4512; 35. Biochemistry (1990) 29:5103; 36. J Biol Chem (1998) 273:11991; 37. J Biol Chem (1994) 269:24596; 38. Anal Biochem (1992) 205:166; 39. J Biol Chem (1996) 271:28399; 40. J Biol Chem (1994) 269:22907; 41. J Chromatogr B Biomed Appl (1994) 659:139; 42. J Chromatogr B Biomed Appl (1994) 659:85; 43. J Chromatogr B Analyt Technol Biomed Life Sci (2006) 831:63; 44. J Lipid Res (1997) 38:1913; 45. Anal Chem (1988) 60:2067; 46. J Chromatogr (1988) 456:421; 47. Tetrahedron Lett (1987) 28:679; 48. J Biol Chem (1988) 263:5724; 49. Biomed Chromatogr (1996) 10:193; 50. J Biomed Mater Res A (2007) 83:1176; 51. J Biol Chem (1997) 272:17444; 52. J Biol Chem (1996) 271:19964; 53. J Org Chem (1981) 46:1666; 54. Anal Chem (1987) 59:1203; 55. J Chromatogr (1990) 508:133; 56. J Chromatogr B Analyt Technol Biomed Life Sci (2004) 803:267; 57. Tetrahedron Lett (1999) 40:1471; 58. Biochemistry (1982) 21:3950.







DATA TABLE 3.4 DERIVATIZATION REAGENTS FOR CARBOXYLIC ACIDS AND CARBOXAMIDESCat. No. MW Storage Soluble Abs EC Em Solvent NotesA91 288.30 L pH >10, DMF 335 5900 493 pH 8A191 175.19 L DMF, DMSO 351 18,000 430 MeOH 1A1318 514.62 F,D,L DMF, EtOH 544 78,000 571 MeOHA1339 491.57 L H2O 425 12,000 532 H2OA1340 533.65 L H2O 426 11,000 531 H2OA1351 397.81 L pH >6, DMF 492 80,000 516 pH 9 2A1353 397.81 L pH >6, DMF 492 68,000 516 pH 9 2A1363 404.38 L pH >6, DMF 491 80,000 515 pH 9 2A1400 218.26 FF,D,L DMF, MeCN 364 6100 411 MeOHA10466 653.38 D,L pH >6, DMF 493 82,000 517 pH 9 2A30674 397.45 F,D,L H2O 353 20,000 437 MeOHA30675 666.58 F,D,L H2O 399 29,000 422 H2O 3A30676 640.61 F,D,L H2O 493 73,000 516 pH 7 4, 5A30677 ~950 F,D,L H2O 555 155,000 572 MeOHA30678 806.94 F,D,L H2O 588 105,000 612 pH 7A30679 ~1000 F,D,L H2O 651 245,000 672 MeOHA30680 812.95 F,D,L H2O 578 93,000 602 pH 7B1355 425.23 F,D,L pH >6, DMF 492 81,000 515 pH 9B2103 341.00 F,D,L DMSO, MeCN 533 62,000 561 CHCl3

B6057 292.17 F,L DMF, MeCN 387 21,000 520 MeOHB30633 207.23 F,D,L DMSO 375 6000 458 MeOHC621 624.49 L H2O 399 30,000 423 H2O 3D112 293.38 L EtOH, DMF 335 4600 526 MeOHD113 335.46 L EtOH, DMF 335 4600 518 MeOHD2390 370.64 F,D,L DMSO, MeCN 503 76,000 510 MeOH 6D6251 544.85 F,D,L DMSO, MeCN 588 64,000 616 MeOH 6D10460 386.47 L DMF, DMSO 373 23,000 571 MeOH 7E2247 191.70 F,D H2O <300 noneH2249 217.13 D H2O <300 noneL2424 600.75 L DMF, DMSO 561 122,000 581 MeOHM6248 174.24 D,A DMF, MeCN <300 noneN2461 373.30 FF,DD,L DMF, CHCl3 260 59,000 395 MeOHO10465 496.47 F,D,L pH >6, DMF 494 75,000 521 pH 9 8P1405 242.28 FF,L DMF, MeCN 340 41,000 375 MeOHQ10464 814.87 L DMSO 560 92,000 none MeOHS10490 268.11 D H2O <300 noneT2425 690.87 L DMF 591 85,000 612 pH 9For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. A191 in aqueous solution (pH 7.0): Abs = 342 nm (EC = 16,000 cm–1M–1), Em = 441 nm.2. Absorption and �uorescence of �uorescein derivatives are pH dependent. Extinction coe�cients and �uorescence quantum yields decrease markedly at pH <7.3. The Alexa Fluor® 405 and Cascade Blue® dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.4. The �uorescence lifetime (τ) of the Alexa Fluor® 488 dye in pH 7.4 bu�er at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.5. Abs and Em of the Alexa Fluor® 488 dye are red-shifted by as much as 16 nm and 25 nm respectively on microarrays relative to aqueous solution values. The magnitude of the spectral shift

depends on the array substrate material. (Biotechniques (2005) 38:127)6. The absorption and �uorescence spectra of BODIPY® derivatives are relatively insensitive to the solvent.7. Fluorescence emission spectrum shifts to shorter wavelengths in nonpolar solvents.8. Absorption and �uorescence of Oregon Green® 488 derivatives are pH dependent only in moderately acidic solutions (pH <5).








PRODUCT LIST 3.4 DERIVATIZATION REAGENTS FOR CARBOXYLIC ACIDS AND CARBOXAMIDESCat. No. Product QuantityA30674 Alexa Fluor® 350 cadaverine 1 mgA30675 Alexa Fluor® 405 cadaverine, trisodium salt4 1 mgA30676 Alexa Fluor® 488 cadaverine, sodium salt 1 mgA30677 Alexa Fluor® 555 cadaverine, disodium salt 1 mgA30680 Alexa Fluor® 568 cadaverine, diammonium salt 1 mgA30678 Alexa Fluor® 594 cadaverine 1 mgA30679 Alexa Fluor® 647 cadaverine, disodium salt 1 mgA1363 5-(aminoacetamido)�uorescein (�uoresceinyl glycine amide) 10 mgA1339 N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt (lucifer yellow ethylenediamine) 25 mgA91 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, sodium salt (EDANS) 1 gA191 7-amino-4-methylcoumarin *reference standard* 100 mgA1351 4’-(aminomethyl)�uorescein, hydrochloride 25 mgA1353 5-(aminomethyl)�uorescein, hydrochloride 10 mgA1318 5-(and-6)-((N-(5-aminopentyl)amino)carbonyl)tetramethylrhodamine (tetramethylrhodamine cadaverine) *mixed isomers* 10 mgA1340 N-(5-aminopentyl)-4-amino-3,6-disulfo-1,8-naphthalimide, dipotassium salt (lucifer yellow cadaverine) 25 mgA10466 5-((5-aminopentyl)thioureidyl)�uorescein, dihydrobromide salt (�uorescein cadaverine) 25 mgA1400 9-anthryldiazomethane (ADAM) 25 mgB30633 bimane amine 5 mgB2103 BODIPY® 493/503 methyl bromide (8-bromomethyl-4,4-di�uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) 5 mgB6057 6-bromoacetyl-2-dimethylaminonaphthalene (badan) 10 mgB1355 5-(bromomethyl)�uorescein 10 mgC621 Cascade Blue® ethylenediamine, trisodium salt 10 mgD10460 Dapoxyl® (2-aminoethyl)sulfonamide 10 mgD2390 4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY® FL EDA) 5 mgD6251 5-(((4-(4,4-di�uoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)pentylamine, hydrochloride (BODIPY® TR cadaverine) 5 mgD112 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide (dansyl ethylenediamine) 100 mgD113 5-dimethylaminonaphthalene-1-(N-(5-aminopentyl))sulfonamide (dansyl cadaverine) 100 mgE2247 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDAC) 100 mgH2249 N-hydroxysulfosuccinimide, sodium salt (NHSS) 100 mgL2424 Lissamine rhodamine B ethylenediamine 10 mgM6248 mono-N-(t-BOC)-propylenediamine 1 gN2461 2-(2,3-naphthalimino)ethyl tri�uoromethanesulfonate 100 mgO10465 Oregon Green® 488 cadaverine *5-isomer* 5 mgP1405 1-pyrenyldiazomethane (PDAM) 25 mgQ10464 QSY® 7 amine, hydrochloride 5 mgS10490 4-sulfo-2,3,5,6-tetra�uorophenol, sodium salt (STP) 100 mgT2425 Texas Red® cadaverine (Texas Red® C5) 5 mg



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