[13]
BIOTIN-CONTAININGREAGENTS
123
[13] B i o t i n - C o n t a i n i n g R e a g e n t s
By MEIR WILCHEK and EDWARD A. BAYER In order to increase the versatility and utility of the avidin-biotin system, a variety of reagents that can label different functional groups on proteins, saccharides, nucleic acids, and other biologically active compounds are required. In addition to promoting the covalent attachment of the biotin moiety to the desired molecule, such reagents can be used for double labeling via biotinylation either of different sites (target, binder, and/or probe) or of different functional groups on the same site. To date, reagents that can label amino groups, thiols, imidazoles and phenols, carboxyls, and aldehydes (carbohydrates), general electrophilic reagents, and photoreactive reagents for indiscriminant labeling have been described in the literature. Figure 1 provides some representative examples of the many reagents currently in use. Although the vast majority of past applications used the N-hydroxysuccinimide ester of biotin (BNHS) for routine biotinylation of proteins, the other reagents are slowly entering the literature for special, interesting, or general applications. In this context, we originally suggested in an earlier review I the use of disulfide- and tartrate-containing derivatives of biotin as cleavable reagents. Indeed, such biotinylating reagents have since been described in the literature, and some are commercially available today. In addition, a new class of reagents, namely, homo- and heterobifunctional, cleavable and noncleavable, biotincontaining cross-linking reagents, is currently being considered for the next generation of biotinylating reagents. Examples of such reagents include maleimido derivatives of either biocytin hydrazide or biocytin-Nhydroxysuccinimide ester for the vectorial cross-linking of thiol groups of one macromolecule with carbohydrate or amino groups of another (Fig. 2). Another such reagent, biotinylglutamic acid dihydrazide, can be used to link two oxidized glycoproteins. These reagents should eventually prove useful for the identification and retrieval of cross-linked proteins using avidin and its derivatives. In this chapter, we describe the synthesis of many of the abovementioned biotin-containing reagents, the original description and/or syntheses of which originated in our laboratory. Although it would have been appropriate to describe the synthesis of a given reagent in the chapter E. A. B a y e r a n d M. W i l c h e k , Methods Biochem. Anal. 26, 1 (1980).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
124
GENERALMETHODOLOGY
-..
0
[13]
0
-..
0
-- B--~-NH-R OI
omines
.-~. B -Bc-o-~>- NO, 0 -~
0 COOH 0 I i1@ B-C-NH CHZ 4-CH-NH-C
COOH 0
l
phenols
0 l
COOH
0
H.
__
N
R
imidozoles
,o, "(CH2)4-C--OH Biohn
R
B- C- NH{CH2)4-C H - N H - C ~ N 2 DBB
0 HNJ~NH
HO N=
o o H II ~---B-C-NH(CHz}4-CH-C-NHN= CH-R
- -
[
ioi i01 - - ~ B-C-NH (CH2}4-CH-C-NHNH2 BCHZ
aldehydes ( sugars ,nuOeic acids )
NH2 COOH 0 I H
corboxyls ( ca r bodii mide- octivoted)
0
-I~ B-e-.H(CHz)4-CH-NH-C-lCHt~) MPB
o
~hio[s
0
o
~
i
NH2 0 0 0 II II II I1. B-C-NH (0H2)4- ICH-C-NHNH-C- R NH2 o. COOH 0 II I II >'~-s- R B - c - NH (CHz)4- CH-NH--C-(CHz)2- oN)fj N. L
0 0 ,,
B - ~ - NH(CH2)5-CNHNHCCH2Br nuc[eophiles
o
,o,
Fs-R ~..-R
B - C-NH (CHzls -CNHN HC-CH-]-O~ef-cR 0
o
B-~-NH-(CHz)3-N--(CH2)~N H - ~ N3 CH5
NO2
phofobiotin
0
B--~-NH-(CHz)fi-N - (CH2)3-NH- ~ nucleic acids, proteins, etc. ( photoillumination )
0"
0
I1~ E}-C-NH-(CH 2 5-C-NH-CH2-CH=CH B-II-dUTP
CH3
0 H o
R
NOz
i01
IlL B-~- NH-(CH2)5- C-NH-- CHz- CH=CH- D N A DNA (nick tronslotion)
OH
FIG. 1. Biotin and examples of some reactive derivatives. B represents the biotin moiety (without the carboxyl group); R represents a biologically active compound that possesses the designated functional group.
describing its application, we decided to present the syntheses of all the reagents together in order to emphasize the fact that they comprise one of the two major "partners" of the system. 2 Short-Chain Biotinylating Reagents It is not surprising that the first biotinylating reagents were simple derivatives of the biotin molecule. It may, however, be surprising to many researchers to learn that the original synthesis of reactive biotin derivatives was not intended for the currently accepted usage, i.e., a general means for the biotinylation of macromolecules. The synthesis of biotin 2 The detailed use of these reagents for the biotinylation of proteins is described elsewhere in this volume (E. A. Bayer and M. Wilchek [14]).
[13]
125
B I O T I N - C O N T A I N I N G REAGENTS
O II /C,, HN NH
0 II
/c\ HN NH
~"
~IL" (CH2)4-C=0 N H I
I
~______~,(CH ~_ 2)2
(ICHz)4 0 / ~ - C O - N H -CH- CO0- N)fJ
0
(CHz)4--C=0 I NH
- (CHzlz-CO-NH-CH-CO-NHNHz 0
0
5-(N-Maleimido propionyl)biocytin N- hydroxysuccinimideester
3-(N-Maleimido propionyl) biocytin hydrazide
0 II /C \ HN NH
~-~ (CH2}4-C=0 I N H I HzNHN- OC- (CHz)z-CH- CO-NH N H z
Biotinyl glutamic acid dihydrazide FIG. 2. Structures of some biotin-containing, bifunctional reagents referred to in the text.
hydrazide was reported as early as 19423 in one of the series of articles that led to the original determination of the structure of biotin. The p-nitrophenyl and N-hydroxysuccinimide esters of biotin (BNP and BNHS, respectively) were also synthesized several years before the era of avidin-biotin technology.4 These reagents were originally designed as potential affinity labels for the site-directed inhibition of the biotin transport system in yeast; BNP successfully inhibited the transport of biotin in these cells, but BNHS did not. It is thus intriguing to note that BNHS, the basic biotinylating reagent for avidin-biotin technology, was originally introduced into the literature as a "negative control" for an entirely different purpose. 3 K. Hofmann, D. B. Melville, and V. du Vigneaud, J. Biol. Chem. 144, 513 (1942). 4 j. M. Becker, M. Wilchek, and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971).
126
GENERAL METHODOLOGY
[13]
Preparation of Biotinyl-N-hydroxysuccinimide Ester (BNHS) 5 Reagents Biotin (Sigma Chemical Co., St. Louis, MO), 10 g N - H y d r o x y s u c c i n i m i d e 6 (Sigma), 6 g Dicyclohexylcarbodiimide (Fluka AG, Buchs, Switzerland), 8 g Dimethylformamide Distilled water Ether 2-Propanol Procedure. Dissolve the biotin in 100 ml of hot dimethylformamide, allow to cool to r o o m temperature, and add the N - h y d r o x y s u c c i n i m i d e with stirring. In a separate flask, dissolve the dicyclohexylcarbodiimide in 20 ml of d i m e t h y l f o r m a m i d e and add to the other reactants. The suspension is stirred overnight at r o o m temperature, and the dicyclohexylurea is filtered. The filtrate is e v a p o r a t e d in vacuo to a minimum volume, and the residue is precipitated with ether, filtered, and washed well with the same solvent. The product is washed further with 2-propanol. Yield, about 10 g (70%). T L C of the product on silica plates shows a single spot, Rf 0.7 [ c h l o r o f o r m : m e t h a n o l (1:2), using dimethylaminocinnamaldehyde spray], 7 c o m p a r e d with R~ 0.3 for biotin using the same system.
Preparation of Biotinyl-p-nitrophenyl Ester (BNP) Reagents Biotin (Sigma), 10 g p - N i t r o p h e n o l (Sigma), 8 g Dicyclohexylcarbodiimide (Fiuka), 8.8 g Dichloromethane Ether 2-Propanol Procedure. The biotin is suspended in 120 ml of dichloromethane at r o o m temperature. To the suspension, the p-nitrophenol and dicyclohexylcarbodiimide are added successively. The flask is covered, and the suspension is stirred overnight at r o o m temperature. The precipitate is 5 In addition to being prepared by the procedure presented in this chapter, BNHS is now available from dozens of companies. Several companies also offer long-chain homologs of BNHS. 6 The N-hydroxysulfosuccinimide ester and other active esters (e.g., N-hydroxyphthalimido and p-nitrophenyl esters) of the above compound may be prepared in a similar manner (with equimolar substitution of the required reactant). 7 D. B. McCormick and J. A. Roth, this series, Vol. 18 [63].
[13]
BIOTIN-CONTAININGREAGENTS
127
filtered, and the filtrate is dried under reduced pressure. The yellow gummy residue is washed well with ether, and the product is recrystallized with 2-propanol. The crystals are washed with ether and dried over sulfuric acid in a desiccator. Yield, 8.8 g (60%); mp 156-158 °. Hydrolysis of the product in 0.2 N NaOH yields an equivalent of p-nitrophenol (determined by A420). TLC on silica plates shows a single spot, Rf 0.45, chloroform :methanol (37: 3), using dimethylaminocinnamaldehyde 7 or NaOH spray.
Preparation of Biotin Hydrazide (BHZ) Reagents Biotin (Sigma), 10 g Hydrazine hydrate Thionyl chloride (Fluka) Methanol Dimethylformamide Procedure. To a chilled solution of methanol (100 ml), thionyl chloride (10 ml) is added. The biotin is then added, and the reaction is allowed to continue overnight at room temperature. The solvent is evaporated to dryness; methanol (100 ml) is again added and reevaporated. The residue is dissolved in 50 ml of methanol, and hydrazine hydrate (12 ml) is added. The reaction is allowed to proceed overnight at room temperature, and the product is filtered and washed with ether. The samples are recrystallized from dimethylformamide. A second crop can be obtained from the concentrated filtrate and recrystallized from dimethylformamide. Total yield, 8.2 g (78%).
Long-Chain Biotin-Containing Intermediates In recent years, it has become apparent that, for steric reasons, the interaction between avidin and a biotinylated macromolecule can be dramatically improved by extending the spacer arm which links the biotin moiety to the surface of the biomolecule. Two derivatives of biotin have found widespread use as extended intermediates for further derivatization into reactive group-specific biotinylating reagents. These are biocytin and biotinyl-N-e-aminocaproic acid. Biocytin (N-e-biotinyl-L-lysine) is a naturally occurring breakdown product of biotin-requiring enzymes. Its synthesis was first described in this connection in 1952. 8 The synthesis of the 8 D. E. Wolf, J. Valiant, R. L. Peck, and K. Folkers, J. Am. Chem. Soc. 74, 2002 (1952).
128
GENERALMETHODOLOGY
[13]
aminocaproic acid derivative of biotin was also described before the advent of avidin-biotin technology.9
Preparation of Biocytin 1oHydrochloride Reagents BNHS, 6.5 g L-Lysine hydrochloride, 3.5 g Cupric carbonate, 12 g Dimethylformamide Sodium bicarbonate, 400 mg Distilled water Ethanol Ether 0.1 N HCI, 10 ml Hydrogen sulfide Procedure. L-Lysine hydrochloride is dissolved in 100 ml of distilled water, and cupric carbonate is added. The solution is boiled in a beaker for 5 min, and the precipitate is filtered. The blue filtrate is cooled in an ice bath, and BNHS (dissolved in 50 ml dimethylformamide) is added. Sodium bicarbonate crystals are introduced to the solution, and the reaction is allowed to continue for 4 hr over ice. The blue precipitate is centrifuged, and the supernatant fluids are decanted. The product is washed successively with water and ethanol and dried with ether. Yield, 7 g (90%). The blue powder is dissolved in 0.1 N HCI, and HzS is bubbled into the solution until no further precipitation occurs. The mixture is heated briefly in a hood until the CuS solid "coagulates." The precipitate is filtered, the residual HzS is evaporated, and the resultant clear solution is lyophilized. Yield, 6 g (75%). Amino acid analysis of the hydrolyzed product yields an equivalent of lysine. TLC on silica gel gives a single spot, R~ 0.2, butanol:acetic acid : water (4 : 1 : 1), with no visible contaminants using ninhydrin or dimethylaminocinnamaldehyde spray. 7
Preparation of Biotinyl-N-e-aminocaproic Acid Reagents BNHS, 10 g e-Aminocaproic acid (Merck, Darmstadt, FRG), 5 g in 60 ml of 0.1 M sodium bicarbonate 9 E. A. Bayer, T. Viswanatha, and M. Wilchek, FEBS Lett. 60, 309 (1975). l0 Biocytin is now also available commercially from Sigma and Calbiochem.
[13]
BIOTIN-CONTAININGREAGENTS
129
D i m e t h y l f o r m a m i d e , 60 ml HCI, 1 and 0. ! N solutions Ether Procedure. Dissolve B N H S in hot dimethylformamide and cool to r o o m temperature. Add dropwise to a bicarbonate solution of e-aminocaproic acid with vigorous magnetic stirring, and continue stirring for 4 hr at r o o m temperature. The solution is acidified with 1 N HC1; the precipitate is washed with cold 0.1 N HC1 and dried with ether. The crystals are dried in vacuo o v e r N a O H . Yield, 7.8 g (74%). T L C on silica gel gives a single spot, Rf 0.5, chloroform : methanol : acetic acid (9: 1: 1), which is easily distinguished from biotin and the reactants ( B N H S Rf 0.8, biotin Rf 0.7, e-aminocaproic acid Rf 0.15). There are no visible contaminants using ninhydrin and biotin-specific 7 sprays. Long-Chain Biotinylating Reagents Owing to the i m p r o v e d interaction between avidin and biotinylated m a c r o m o l e c u l e s which bear extended spacer arms, there has been a recent trend to use long-chain derivatives of biotin as reagents for incorporating the biotin moiety into the binder of choice. Of the two intermediates used for synthesis of the long-chain reagents, biocytin is preferred where applicable H since the product is generally more soluble in aqueous solutions b e c a u s e of the free amino or carboxyl group that remains after derivatization.
Preparation of Biotinyl-N-e-aminocaproyl-N-hydroxysuccinimide Ester (BcapNHS) Reagents B i o t i n y l - N - e - a m i n o c a p r o i c acid, 3.6 g N - H y d r o x y s u c c i n i m i d e 6 (Sigma), 1.4 g Dicyclohexylcarbodiimide (Fluka), 2.3 g N-Methylpyrrolidone Ether Procedure. Dissolve b i o t i n y l - N - e - a m i n o c a p r o i c acid in 70 ml of N - m e t h y l p y r r o l i d o n e . lz Add the N - h y d r o x y s u c c i n i m i d e followed by the 11One should always keep in mind that the remaining group of the biocytin derivative is not necessarily "innocent." Thus, N-hydroxysuccinimide derivatives of biocytin cannot be synthesized, since the free amino group would react with the active ester. In another example, the pK of biocytin hydrazide is substantially higher than that of biocytin; this may, in some cases, affect its performance as a biotinylating reagent. 12N-Methylpyrrolidone is more suitable than dimethylformamide as a solvent in this reaction. In order to facilitate the solubilization, warm the solvent to 80°, then cool immediately to room temperature.
130
GENERALMETHODOLOGY
[13]
dicyclohexylcarbodiimide, and stir gently overnight at room temperature. After filtering the dicyclohexylurea which forms, the product is precipitated by ether (-500 ml) and collected on a sintered glass funnel. Rinse thoroughly with ether and dry in a vacuum desiccator over phosphorus pentoxide. Yield, 3.2 g (70%). TLC on silica gel gives a single spot, Rf 0.3, chloroform : methanol : acetic acid (17 : 2 : I).
Preparation of Biotinyl-N-e-aminocaproyl-p-nitrophenyl Ester (BcapNP) Reagents Biotinyl-N-e-aminocaproic acid, 1 g p-Nitrophenol (Fluka), 0.6 g Dicyclohexylcarbodiimide (Fluka), 0.62 g N-Methylpyrrolidone Ether Procedure. Dissolve biotinyl-N-e-aminocaproic acid in 20 ml Nmethylpyrrolidone, j2 Add the p-nitrophenol followed by the dicyclohexylcarbodiimide and stir gently overnight at room temperature. Filter off the dicyclohexylurea which forms. The product is precipitated by ether and collected on a sintered glass funnel. Rinse thoroughly with ether and dry in a vacuum desiccator.
Preparation of Biocytin Hydrazide (BCHZ) Reagents Biocytin, 1 g Hydrazine hydrate (Merck) Thionyl chloride (Fluka) Methanol Ether Dimethylformamide Distilled water Porapak Type Q column (Waters Associates Inc., Milford, MA), 10× I c m Procedure. Thionyl chloride (2.5 ml) is added to a solution of methanol (25 ml) cooled in an ice water-acetone bath. Biocytin (1 g) is then added, and the mixture is allowed to react overnight with constant stirring. The solvent is reduced to a minimal volume, and ether is added. The precipitate is filtered, washed with ether, and dried over NaOH under reduced pressure. TLC of the product (biocytin methyl ester) on silica gel gives one spot [Rf 0.48, butanol : acetic acid : water (4 : 1 : 1)] with no visible
[13]
BIOTIN-CONTAININGREAGENTS
131
contaminants using dimethylaminocinnamaldehyde 7 and ninhydrin sprays. The methyl ester of biocytin (1 g) is dissolved in 20 ml of methanol, and hydrazine hydrate (1 ml) is added. After 48 hr at room temperature, the solvent is concentrated to dryness; drying is continued further in a desiccator under reduced pressure in the presence of H 2 8 0 4 until the odor of hydrazine can no longer be detected. The product is dissolved in water (10 ml) and passed through a column containing Porapak Type Q. The column is eluted with water, and 10-ml fractions are collected.13 Fractions containing biocytin hydrazide are detected on TLC ( R f 0. I 1, using methanol as the solvent system) by dimethylaminocinnamaldehyde 7 and ninhydrin sprays. TM The fractions containing the product are pooled, concentrated to dryness, dissolved in a minimal amount of methanol, and precipitated with ether. Biocytin hydrazide can be crystallized from hot ethanol. On reaction with acetone, three reaction products are obtained (TLC, using methanol as solvent) with R f values of 0.24, 0.55, and 0.65, representing the anticipated reaction products. We have also prepared the same compound from t-Boc biocytin 15and tert-butyl carbazate in the presence of dicyclohexylcarbodiimide in a dimethylformamide solution. The protecting groups are removed with 4 N HCI in dioxane.
Preparation of 3-(N-Maleimidopropionyl)biocytin (MPB)16 Reagents Biocytin hydrochloride, I g in 10 ml of 0.5 M sodium bicarbonate 3-(N-Maleimidopropionyl)-N-hydroxysuccinimide ester (Calbiochem, San Diego, CA), 0.7 g Dimethylformamide 1 N HC1 Ether Procedure. 3-(N-Maleimidopropionyl)-N-hydroxysuccinimide ester is dissolved in dimethylformamide (10 ml) and added dropwise to the biocy13 The first several fractions usually contain residual hydrazine hydrochloride. It is essential to remove all of the latter since hydrazine reacts well with aldehydes and, as an impurity, would reduce the final efficiency of the hydrazide reagent. t4 To hasten elution of the hydrazide, the column is eluted with 80% aqueous methanol following the hydrazine peak. The fractions are then eluted as a sharp peak. ~5E. Bayer and M. Wilchek, this series, Vol. 34 [20]. 16 MPB and similar maleimido analogs of biocytin are now available from Sigma and Calbiochem.
132
GENERALMETHODOLOGY
[13]
tin solution. The reaction is carried out for 2 hr, after which the solution is acidified with 1 N HCI. The product is allowed to crystallize at 4°. The crystals are washed with cold 0.1 N HC1 and then dried with ether. Yield, 0.8 g (60%). MPB appears on TLC as a single spot, Rf 0.3, chloroform : methanol (1 : 1). The product can be detected by dimethylaminocinnamaldehyde,7 iodine vapors, or by burning the TLC plate. In order to test the reactivity of the spot to sulfhydryls, a 5-fold molar excess of dithiothreitol is interacted with MPB. The free sulfhydryl-containing derivative, MPB-SH, is formed. Unreacted dithiothreitol (Rf 0.9) is separated from the sulfhydryl adduct (Rf 0.35) using the above TLC system. Both dithiothreitol and MPB-SH are detected using 5,5'-dithiobis(2-nitrobenzoic acid) spray (0.1% in ethanol :0.1% NaHCO3, 1:1, v/v). Only the biotin-containing derivative is visible using dimethylaminocinnamaldehyde spray. 7
Preparation of Other Maleimidobiocytin Derivatives Other maleimido derivatives of biocytin can be prepared similarly, using commercially available maleimido aliphatic or aromatic N-hydroxysuccinimide esters. The aromatic derivatives are less soluble and hence less effective. 4-(N-Maleimidobutyryl)-N-hydroxysuccinimide ester and 6-(N-maleimidocaproyl)-N-hydroxysuccinimide ester are commercially available (Calbiochem, Sigma). The biocytin derivatives prepared from these compounds behave similarly to MPB.
Preparation of p-Aminobenzoylbiocytin N-t-Boc-p-aminobenzoic Acid Reagents p-Aminobenzoic acid (Fluka), 13.7 g BOC-ON (Fluka), 20 g 1 N NaOH Dioxane Petroleum ether Distilled water 10% Citric acid Procedure. p-Aminobenzoic acid is dissolved in 1 N NaOH (120 ml), and BOC-ON (dissolved in 50 ml dioxane) is added. The reaction mixture is stirred vigorously overnight. Water is added, and the solution is extracted with petroleum ether to remove the dioxane. The aqueous layer is acidified with citric acid, and the precipitate is collected, washed with
[13]
BIOTIN-CONTAININGREAGENTS
133
water, and air dried. Yield, 15 g (65%); mp 185°. TLC on silica plates, ether:petroleum ether (17: 3), yields a single spot (Rf 0.44) which could be distinguished from the starting material (Rf 0.40) by its insensitivity to ninhydrin staining.
t-Boc-p-aminobenzoyl-N-hydroxysuccinimide Ester Reagents N-t-Boc-p-aminobenzoic acid (6 g) N-Hydroxysuccinimide (Sigma), 3 g Dicyclohexylcarbodiimide (Fluka), 5.5 g Dioxane Ether Procedure. To the N-hydroxysuccinimide in dioxane (100 ml), solid N-t-Boc-p-aminobenzoic acid is added. The dicyclohexylcarbodiimide (dissolved in 20 ml dioxane) is added, and the solution is stirred for I hr. The filtrate is concentrated to minimal volume, and the product is crystallized from ether. Yield, 6.5 g (78%); mp 165°. TLC, ether:petroleum ether (19: 1), gives a single spot with gf 0.47 compared to 0.57 for the starting material.
t-Boc-p-aminobenzoylbiocytin Reagents Biocytin hydrochloride, 1 g, dissolved in 11 ml of 0.5 N NaOH t-Boc-p-aminobenzoyl-N-hydroxysuccinimide ester, 0.85 g Dimethylformamide Ether Petroleum ether Distilled water 10% Citric acid Procedure. t-Boc-p-aminobenzoyl-N-hydroxysuccinimide ester is dissolved in 10 ml of dimethylformamide, and biocytin is added. Additional dimethylformamide is added if a precipitate is formed, and the reaction is carried out overnight at room temperature. Water is then added, and the reaction mixture is extracted successively with ether and petroleum ether in order to remove the dimethylformamide. The solution is acidified with citric acid, and the precipitate is collected by filtration. The precipitate is washed with ether to remove traces of the reactants. Yield, 1.2 g (85%); mp 171°. TLC, butanol : acetic acid : water (4 : 1 : 1), gives a single spot with an Rf of 0.6 compared to 0.14 for biocytin.
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GENERALMETHODOLOGY
[13]
p-Aminobenzoylbiocytin (TFA salt) Reagents t-Boc-p-aminobenzoylbiocytin, 1.2 g Trifluoroacetic acid (TFA) Dichloromethane Ether Procedure. t-Boc-p-Aminobenzoylbiocytin is dissolved in 20 ml of a 1 : 1 mixture of TFA and dichloromethane and left at room temperature for 30 min. Ether is added, and the precipitate is filtered and washed with ether until dry. Yield, 1.05 g (88%). TLC, butanol:acetic acid:water (4 : 1 : 1), gives a single spot ( R f 0.4). On reaction with NaNO2, the product reacts with tyrosine.
p-Diazobenzoylbiocytin (DBB) 17 Reagents p-Aminobenzoylbiocytin, 2 mg 2 N HCi NaNOz, 7.7 mg/ml (ice-cold aqueous solution) 1 N NaOH 0.1 M Borate buffer, pH 8.4 Procedure. p-Aminobenzoylbiocytin is dissolved in ice-cold 2 N HCI (40.7/zl). An equivalent volume of NaNO2 is added, and the reaction is allowed to continue for 5 min at 4°. The reaction is terminated on addition of 1 N NaOH (35/zl). The DBB solution is brought to the desired concentration by dilution with borate buffer, and the solution is used immediately. Biotin-Containing Cross-Linking Reagents A new class of probes includes a series of trifunctional reagents in the general form B X--C--Y
where B includes the bicyclic ring system of biotin, C is a bifurcated spacer arm, and X and Y are reactive functional groups. The latter groupspecific functions can either be homofunctional (e.g., two N-hydroxysuccinimide groups) or heterofunctional (e.g., a maleimide and hydrazide 17 Diazobenzoyl derivatives are chemically unstable and cannot be stored for long periods of time. The preparation of DBB from the aminobenzoyl derivative is therefore performed immediately prior to use. The precursor is now available commercially from Calbiochem.
[13]
BIOTIN-CONTAININGREAGENTS
135
group). The spacer may be extended in any direction by addition of o~amino acids, in order to facilitate vectorial interaction with target molecules or with the avidin-containing probe. The spacer either can be uncleavable or can theoretically be cleavable in one, two, or three directions; the number and site(s) of cleavage on a biotinylated crosslinking reagent would be determined by the ultimate goal of the researcher, i.e., whether cleawtge of the biotin moiety is required to facilitate isolation (release from an avidin column) or whether cleavage of cross-linked macromolecules is desired for identification purposes (e.g., on two-dimensional SDS-PAGE). Suitable spacers may include mono-, di-, oligo-, and polysaccharides and their substituted derivatives. Alternatively, certain synthetic and naturally occurring amino acids (e.g., lysine, ornithine, aspartic and glutamic acids), dipeptides, and oligopeptides are also suitable as bifurcated spacers. Tartryl (periodate-cleavable) and disulfide (thiol-cleavable) derivatives can be used as cleavable extender arms. A wide variety of such reagents can be designed, some of which are presented in Fig. 3. The bifurcated biotin-containing reagents can be used for preparing conjugates of two different protein species, such as a monoclonal antibody and an enzyme. Alternatively, these reagents can be used for combined cross-linkage and biotinylation of molecular components of a complex biological system, such as the cell membrane. The presence of the biotin moiety allows subsequent isolation, identification, or targeting of the cross-linked molecules of interest. Some examples in the use of these reagents are presented later in this volume. 2
Preparation of 3-(N-Maleimidopropionyl)biocytin N-Hydroxysuccinimide Ester Reagents MPB, 523 mg N-Hydroxysuccinimide,6 125 mg Dicyclohexylcarbodiimide, 225 mg Dimethylformamide Ether Procedure. 3-(N-Maleimidopropionyl)biocytin is dissolved in 5 ml of dimethylformamide, and the N-hydroxysuccinimide is added. The solution is cooled to 4°, and dicyclohexylcarbodiimide is added. The reaction mixture is maintained for 24 hr at 4°. The dicyciohexylurea precipitate is filtered and discarded, and the product is precipitated directly from the filtrate with ether. The precipitate is collected, washed with ether, and the amount of N-hydroxysuccinimide ester is determined spectrophotometrically (/3261 10,000 M 1 cm 1) after hydrolysis with 0.5 M NH4OH.
136
GENERAL METHODOLOGY
0 II / c\ HN NH ~~
[13]
0 II /c\ HN NH
(CH2)4_c= 0 I NH I (CH2)4
~'~(CHz)4_C= 0 I NH X I -OC-(CH 2)2-CH-C0- y
I
X -NH-CH-CO- y
Functional groups
for X and
y
0
Spacers Noncleavable
-0-1~
HOOC-(CHz)n- COOH HzN-(CH2)n-NHz HzN-(CH2)n- COOH
-0"-~'-
NOz
HzN- (CHz)4-CH-COOH
o
%
-C- (CHz)n-N~ I
Cleavable
o
HOOC-[CHz)2-S-S- (CHz)2-COOH
-NHNH z 0 II /-~-~ + -C ~-4~.~N z 0 /-~'~ + -CII~-'~,..~--N~
or
or
HzN-
- N H - - ~ - - N;
-NH-~
N;
{CH2]2 - $ - $ - (C H2)2-
NH2
HOOC-CH- CHz- S-S-CH2-CH-COOH HO OH I I HOOC-CH-CH-COOH OH OH I I HOOC-(CHz)n-HNOC-CH-CH-CONH-(CHz)n-COOH
FIG. 3. Examples of biotin-containing cross-linking reagents. The examples shown are based either on biocytin, which provides both an amine and a carboxyl group for further derivatization with reactive groups X and Y, or on biotinyl glutamate, which provides two free carboxyl groups for the same purpose. The reactive functional groups X and Y can either be identical (in the case of homobifunctional reagents) or different (heterobifunctional reagents). In some cases, a given functional group is incompatible with another (e.g., a hydrazide derivative on one side and a p-nitrophenyl or N-hydroxysuccinimide group on the other), since the reagent would polymerize. The reactive groups and/or the biotin moiety can be extended further from the point of bifurcation by the incorporation of a suitable spacer group. If the latter is a cystinyl or tartryl derivative, the resultant reagent would be cleavable at the corresponding position.
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137
Preparation of 3-(N-Maleimidopropionyl)biocytin Hydrazide Reagents 3-(N-Maleimidopropionyl)biocytin N-hydroxysuccinimide ester, 125 mg in 1 ml of dimethylformamide Hydrazine hydrate, 10/.d in 1 ml of 0.5 M sodium bicarbonate Methanol Ether Procedure. The solution of 3-(N-maleimidopropionyl)biocytin Nhydroxysuccinimide is added dropwise to the hydrazine hydrate solution. The reaction is carried out for 3 hr at room temperature. The solution is concentrated to dryness, dissolved in a minimal amount of methanol, and the product is precipitated with ether. Purity is determined by TLC, and the product is identified by its reaction with acetone (and consequent change in Rf value). Other hydrazide derivatives bearing long spacer arms are prepared by substituting 3 to 5 molar equivalents of the respective dihydrazide (e.g., 100-200 mg adipic acid dihydrazide). These derivatives can also be prepared directly from 3-(N-maleimidopropionyl)biocytin using either hydrazine or a desired dihydrazide derivative in the presence of dicyclohexylcarbodiimide.
Biotinyl-L-glutamic Acid Reagents BNHS, 3.4 g in 30 ml of dimethylformamide L-Glutamic acid, 1.5 g in 30 ml of 0.5 M sodium bicarbonate l N HCI Distilled water Procedure. The BNHS and glutamic acid solutions are mixed, and the reaction is carried out for 2 hr at room temperature. The solution is acidified with HC]; the resultant precipitate is washed with cold water and dried in vacuo. Other aminodicarboxylic acids, peptides, etc., are prepared by substituting equimolar amounts for glutamic acid.
Biotinyl-L-glutamic Acid Dihydrazide Reagents Biotinyl-L-glutamic acid, 1 g in 10 ml of methanol Thionyl chloride, 1 ml Methanol Sodium hydroxide
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Hydrazine hydrate, l ml 2-Propanol Procedure. The thionyl chloride and the methanolic solution of biotinyl-L-glutamic acid are cooled separately in an acetone-dry ice bath before mixing. The reaction is left at room temperature for 24 hr. The methanol is removed under vacuum; an equivalent volume of methanol is added and removed again by evaporation, and this step is repeated twice again. The residue is dried in a desiccator over sodium hydroxide (to remove traces of HCI). The residue is again dissolved in methanol, hydrazine hydrate is added, and the reaction is left at room temperature for 24 hr. A portion of the biotinylglutamic acid dihydrazide precipitates out and is filtered. The remainder is obtained by evaporation of the methanol and crystallization from 2-propanol.
[14] P r o t e i n B i o t i n y l a t i o n
By EDWARD A. BAYER and MEIR WILCHEK An essential prerequisite in the successful application of avidin-biotin technology is the incorporation of the biotin moiety into the experimental system. This may be accomplished by direct biotinylation of the target system using one of the many examples of group-specific biotin-containing reagents described elsewhere in this volume. J However, another alternative, one that contributes toward the virtually unlimited potential of the system, is the capacity to biotinylate biologically active molecules which maintain their recognition properties toward a given target molecule? -4 In this case, biotin may be introduced chemically via a variety of functional groups on the binder using the same group-specific reagents. In this chapter, we present some of the procedures by which we have incorporated biotin moieties into proteins. Although in earlier w o r k s 4,5 w e have professed that biotinylation of a macromolecule is relatively harmless to its activity and only minimally affects its physical and chemical properties, this is not necessarily true, and a given biotinylated binder should always be thoroughly characterized prior to use. We therefore M. 2 E. 3 E. 4 E. 5 M.
Wilchek and E. A. Bayer, this volume [131. A. Bayer, M. Wilchek, and E. Skutelsky, F E B S Lett. 68, 240 (1976). A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978). A. Bayer and M. Wilchek, M e t h o d s Biochem. Anal. 26, 1 (1980). Wilchek and E. A. Bayer, l m m u n o l . Today 5, 39 (1984).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 184
Copyright .~; 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOTIN-CONTAININGREAGENTS
123
[13] B i o t i n - C o n t a i n i n g R e a g e n t s
By MEIR WILCHEK and EDWARD A. BAYER In order to increase the versatility and utility of the avidin-biotin system, a variety of reagents that can label different functional groups on proteins, saccharides, nucleic acids, and other biologically active compounds are required. In addition to promoting the covalent attachment of the biotin moiety to the desired molecule, such reagents can be used for double labeling via biotinylation either of different sites (target, binder, and/or probe) or of different functional groups on the same site. To date, reagents that can label amino groups, thiols, imidazoles and phenols, carboxyls, and aldehydes (carbohydrates), general electrophilic reagents, and photoreactive reagents for indiscriminant labeling have been described in the literature. Figure 1 provides some representative examples of the many reagents currently in use. Although the vast majority of past applications used the N-hydroxysuccinimide ester of biotin (BNHS) for routine biotinylation of proteins, the other reagents are slowly entering the literature for special, interesting, or general applications. In this context, we originally suggested in an earlier review I the use of disulfide- and tartrate-containing derivatives of biotin as cleavable reagents. Indeed, such biotinylating reagents have since been described in the literature, and some are commercially available today. In addition, a new class of reagents, namely, homo- and heterobifunctional, cleavable and noncleavable, biotincontaining cross-linking reagents, is currently being considered for the next generation of biotinylating reagents. Examples of such reagents include maleimido derivatives of either biocytin hydrazide or biocytin-Nhydroxysuccinimide ester for the vectorial cross-linking of thiol groups of one macromolecule with carbohydrate or amino groups of another (Fig. 2). Another such reagent, biotinylglutamic acid dihydrazide, can be used to link two oxidized glycoproteins. These reagents should eventually prove useful for the identification and retrieval of cross-linked proteins using avidin and its derivatives. In this chapter, we describe the synthesis of many of the abovementioned biotin-containing reagents, the original description and/or syntheses of which originated in our laboratory. Although it would have been appropriate to describe the synthesis of a given reagent in the chapter E. A. B a y e r a n d M. W i l c h e k , Methods Biochem. Anal. 26, 1 (1980).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
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GENERALMETHODOLOGY
-..
0
[13]
0
-..
0
-- B--~-NH-R OI
omines
.-~. B -Bc-o-~>- NO, 0 -~
0 COOH 0 I i1@ B-C-NH CHZ 4-CH-NH-C
COOH 0
l
phenols
0 l
COOH
0
H.
__
N
R
imidozoles
,o, "(CH2)4-C--OH Biohn
R
B- C- NH{CH2)4-C H - N H - C ~ N 2 DBB
0 HNJ~NH
HO N=
o o H II ~---B-C-NH(CHz}4-CH-C-NHN= CH-R
- -
[
ioi i01 - - ~ B-C-NH (CH2}4-CH-C-NHNH2 BCHZ
aldehydes ( sugars ,nuOeic acids )
NH2 COOH 0 I H
corboxyls ( ca r bodii mide- octivoted)
0
-I~ B-e-.H(CHz)4-CH-NH-C-lCHt~) MPB
o
~hio[s
0
o
~
i
NH2 0 0 0 II II II I1. B-C-NH (0H2)4- ICH-C-NHNH-C- R NH2 o. COOH 0 II I II >'~-s- R B - c - NH (CHz)4- CH-NH--C-(CHz)2- oN)fj N. L
0 0 ,,
B - ~ - NH(CH2)5-CNHNHCCH2Br nuc[eophiles
o
,o,
Fs-R ~..-R
B - C-NH (CHzls -CNHN HC-CH-]-O~ef-cR 0
o
B-~-NH-(CHz)3-N--(CH2)~N H - ~ N3 CH5
NO2
phofobiotin
0
B--~-NH-(CHz)fi-N - (CH2)3-NH- ~ nucleic acids, proteins, etc. ( photoillumination )
0"
0
I1~ E}-C-NH-(CH 2 5-C-NH-CH2-CH=CH B-II-dUTP
CH3
0 H o
R
NOz
i01
IlL B-~- NH-(CH2)5- C-NH-- CHz- CH=CH- D N A DNA (nick tronslotion)
OH
FIG. 1. Biotin and examples of some reactive derivatives. B represents the biotin moiety (without the carboxyl group); R represents a biologically active compound that possesses the designated functional group.
describing its application, we decided to present the syntheses of all the reagents together in order to emphasize the fact that they comprise one of the two major "partners" of the system. 2 Short-Chain Biotinylating Reagents It is not surprising that the first biotinylating reagents were simple derivatives of the biotin molecule. It may, however, be surprising to many researchers to learn that the original synthesis of reactive biotin derivatives was not intended for the currently accepted usage, i.e., a general means for the biotinylation of macromolecules. The synthesis of biotin 2 The detailed use of these reagents for the biotinylation of proteins is described elsewhere in this volume (E. A. Bayer and M. Wilchek [14]).
[13]
125
B I O T I N - C O N T A I N I N G REAGENTS
O II /C,, HN NH
0 II
/c\ HN NH
~"
~IL" (CH2)4-C=0 N H I
I
~______~,(CH ~_ 2)2
(ICHz)4 0 / ~ - C O - N H -CH- CO0- N)fJ
0
(CHz)4--C=0 I NH
- (CHzlz-CO-NH-CH-CO-NHNHz 0
0
5-(N-Maleimido propionyl)biocytin N- hydroxysuccinimideester
3-(N-Maleimido propionyl) biocytin hydrazide
0 II /C \ HN NH
~-~ (CH2}4-C=0 I N H I HzNHN- OC- (CHz)z-CH- CO-NH N H z
Biotinyl glutamic acid dihydrazide FIG. 2. Structures of some biotin-containing, bifunctional reagents referred to in the text.
hydrazide was reported as early as 19423 in one of the series of articles that led to the original determination of the structure of biotin. The p-nitrophenyl and N-hydroxysuccinimide esters of biotin (BNP and BNHS, respectively) were also synthesized several years before the era of avidin-biotin technology.4 These reagents were originally designed as potential affinity labels for the site-directed inhibition of the biotin transport system in yeast; BNP successfully inhibited the transport of biotin in these cells, but BNHS did not. It is thus intriguing to note that BNHS, the basic biotinylating reagent for avidin-biotin technology, was originally introduced into the literature as a "negative control" for an entirely different purpose. 3 K. Hofmann, D. B. Melville, and V. du Vigneaud, J. Biol. Chem. 144, 513 (1942). 4 j. M. Becker, M. Wilchek, and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971).
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Preparation of Biotinyl-N-hydroxysuccinimide Ester (BNHS) 5 Reagents Biotin (Sigma Chemical Co., St. Louis, MO), 10 g N - H y d r o x y s u c c i n i m i d e 6 (Sigma), 6 g Dicyclohexylcarbodiimide (Fluka AG, Buchs, Switzerland), 8 g Dimethylformamide Distilled water Ether 2-Propanol Procedure. Dissolve the biotin in 100 ml of hot dimethylformamide, allow to cool to r o o m temperature, and add the N - h y d r o x y s u c c i n i m i d e with stirring. In a separate flask, dissolve the dicyclohexylcarbodiimide in 20 ml of d i m e t h y l f o r m a m i d e and add to the other reactants. The suspension is stirred overnight at r o o m temperature, and the dicyclohexylurea is filtered. The filtrate is e v a p o r a t e d in vacuo to a minimum volume, and the residue is precipitated with ether, filtered, and washed well with the same solvent. The product is washed further with 2-propanol. Yield, about 10 g (70%). T L C of the product on silica plates shows a single spot, Rf 0.7 [ c h l o r o f o r m : m e t h a n o l (1:2), using dimethylaminocinnamaldehyde spray], 7 c o m p a r e d with R~ 0.3 for biotin using the same system.
Preparation of Biotinyl-p-nitrophenyl Ester (BNP) Reagents Biotin (Sigma), 10 g p - N i t r o p h e n o l (Sigma), 8 g Dicyclohexylcarbodiimide (Fiuka), 8.8 g Dichloromethane Ether 2-Propanol Procedure. The biotin is suspended in 120 ml of dichloromethane at r o o m temperature. To the suspension, the p-nitrophenol and dicyclohexylcarbodiimide are added successively. The flask is covered, and the suspension is stirred overnight at r o o m temperature. The precipitate is 5 In addition to being prepared by the procedure presented in this chapter, BNHS is now available from dozens of companies. Several companies also offer long-chain homologs of BNHS. 6 The N-hydroxysulfosuccinimide ester and other active esters (e.g., N-hydroxyphthalimido and p-nitrophenyl esters) of the above compound may be prepared in a similar manner (with equimolar substitution of the required reactant). 7 D. B. McCormick and J. A. Roth, this series, Vol. 18 [63].
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127
filtered, and the filtrate is dried under reduced pressure. The yellow gummy residue is washed well with ether, and the product is recrystallized with 2-propanol. The crystals are washed with ether and dried over sulfuric acid in a desiccator. Yield, 8.8 g (60%); mp 156-158 °. Hydrolysis of the product in 0.2 N NaOH yields an equivalent of p-nitrophenol (determined by A420). TLC on silica plates shows a single spot, Rf 0.45, chloroform :methanol (37: 3), using dimethylaminocinnamaldehyde 7 or NaOH spray.
Preparation of Biotin Hydrazide (BHZ) Reagents Biotin (Sigma), 10 g Hydrazine hydrate Thionyl chloride (Fluka) Methanol Dimethylformamide Procedure. To a chilled solution of methanol (100 ml), thionyl chloride (10 ml) is added. The biotin is then added, and the reaction is allowed to continue overnight at room temperature. The solvent is evaporated to dryness; methanol (100 ml) is again added and reevaporated. The residue is dissolved in 50 ml of methanol, and hydrazine hydrate (12 ml) is added. The reaction is allowed to proceed overnight at room temperature, and the product is filtered and washed with ether. The samples are recrystallized from dimethylformamide. A second crop can be obtained from the concentrated filtrate and recrystallized from dimethylformamide. Total yield, 8.2 g (78%).
Long-Chain Biotin-Containing Intermediates In recent years, it has become apparent that, for steric reasons, the interaction between avidin and a biotinylated macromolecule can be dramatically improved by extending the spacer arm which links the biotin moiety to the surface of the biomolecule. Two derivatives of biotin have found widespread use as extended intermediates for further derivatization into reactive group-specific biotinylating reagents. These are biocytin and biotinyl-N-e-aminocaproic acid. Biocytin (N-e-biotinyl-L-lysine) is a naturally occurring breakdown product of biotin-requiring enzymes. Its synthesis was first described in this connection in 1952. 8 The synthesis of the 8 D. E. Wolf, J. Valiant, R. L. Peck, and K. Folkers, J. Am. Chem. Soc. 74, 2002 (1952).
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GENERALMETHODOLOGY
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aminocaproic acid derivative of biotin was also described before the advent of avidin-biotin technology.9
Preparation of Biocytin 1oHydrochloride Reagents BNHS, 6.5 g L-Lysine hydrochloride, 3.5 g Cupric carbonate, 12 g Dimethylformamide Sodium bicarbonate, 400 mg Distilled water Ethanol Ether 0.1 N HCI, 10 ml Hydrogen sulfide Procedure. L-Lysine hydrochloride is dissolved in 100 ml of distilled water, and cupric carbonate is added. The solution is boiled in a beaker for 5 min, and the precipitate is filtered. The blue filtrate is cooled in an ice bath, and BNHS (dissolved in 50 ml dimethylformamide) is added. Sodium bicarbonate crystals are introduced to the solution, and the reaction is allowed to continue for 4 hr over ice. The blue precipitate is centrifuged, and the supernatant fluids are decanted. The product is washed successively with water and ethanol and dried with ether. Yield, 7 g (90%). The blue powder is dissolved in 0.1 N HCI, and HzS is bubbled into the solution until no further precipitation occurs. The mixture is heated briefly in a hood until the CuS solid "coagulates." The precipitate is filtered, the residual HzS is evaporated, and the resultant clear solution is lyophilized. Yield, 6 g (75%). Amino acid analysis of the hydrolyzed product yields an equivalent of lysine. TLC on silica gel gives a single spot, R~ 0.2, butanol:acetic acid : water (4 : 1 : 1), with no visible contaminants using ninhydrin or dimethylaminocinnamaldehyde spray. 7
Preparation of Biotinyl-N-e-aminocaproic Acid Reagents BNHS, 10 g e-Aminocaproic acid (Merck, Darmstadt, FRG), 5 g in 60 ml of 0.1 M sodium bicarbonate 9 E. A. Bayer, T. Viswanatha, and M. Wilchek, FEBS Lett. 60, 309 (1975). l0 Biocytin is now also available commercially from Sigma and Calbiochem.
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BIOTIN-CONTAININGREAGENTS
129
D i m e t h y l f o r m a m i d e , 60 ml HCI, 1 and 0. ! N solutions Ether Procedure. Dissolve B N H S in hot dimethylformamide and cool to r o o m temperature. Add dropwise to a bicarbonate solution of e-aminocaproic acid with vigorous magnetic stirring, and continue stirring for 4 hr at r o o m temperature. The solution is acidified with 1 N HC1; the precipitate is washed with cold 0.1 N HC1 and dried with ether. The crystals are dried in vacuo o v e r N a O H . Yield, 7.8 g (74%). T L C on silica gel gives a single spot, Rf 0.5, chloroform : methanol : acetic acid (9: 1: 1), which is easily distinguished from biotin and the reactants ( B N H S Rf 0.8, biotin Rf 0.7, e-aminocaproic acid Rf 0.15). There are no visible contaminants using ninhydrin and biotin-specific 7 sprays. Long-Chain Biotinylating Reagents Owing to the i m p r o v e d interaction between avidin and biotinylated m a c r o m o l e c u l e s which bear extended spacer arms, there has been a recent trend to use long-chain derivatives of biotin as reagents for incorporating the biotin moiety into the binder of choice. Of the two intermediates used for synthesis of the long-chain reagents, biocytin is preferred where applicable H since the product is generally more soluble in aqueous solutions b e c a u s e of the free amino or carboxyl group that remains after derivatization.
Preparation of Biotinyl-N-e-aminocaproyl-N-hydroxysuccinimide Ester (BcapNHS) Reagents B i o t i n y l - N - e - a m i n o c a p r o i c acid, 3.6 g N - H y d r o x y s u c c i n i m i d e 6 (Sigma), 1.4 g Dicyclohexylcarbodiimide (Fluka), 2.3 g N-Methylpyrrolidone Ether Procedure. Dissolve b i o t i n y l - N - e - a m i n o c a p r o i c acid in 70 ml of N - m e t h y l p y r r o l i d o n e . lz Add the N - h y d r o x y s u c c i n i m i d e followed by the 11One should always keep in mind that the remaining group of the biocytin derivative is not necessarily "innocent." Thus, N-hydroxysuccinimide derivatives of biocytin cannot be synthesized, since the free amino group would react with the active ester. In another example, the pK of biocytin hydrazide is substantially higher than that of biocytin; this may, in some cases, affect its performance as a biotinylating reagent. 12N-Methylpyrrolidone is more suitable than dimethylformamide as a solvent in this reaction. In order to facilitate the solubilization, warm the solvent to 80°, then cool immediately to room temperature.
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GENERALMETHODOLOGY
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dicyclohexylcarbodiimide, and stir gently overnight at room temperature. After filtering the dicyclohexylurea which forms, the product is precipitated by ether (-500 ml) and collected on a sintered glass funnel. Rinse thoroughly with ether and dry in a vacuum desiccator over phosphorus pentoxide. Yield, 3.2 g (70%). TLC on silica gel gives a single spot, Rf 0.3, chloroform : methanol : acetic acid (17 : 2 : I).
Preparation of Biotinyl-N-e-aminocaproyl-p-nitrophenyl Ester (BcapNP) Reagents Biotinyl-N-e-aminocaproic acid, 1 g p-Nitrophenol (Fluka), 0.6 g Dicyclohexylcarbodiimide (Fluka), 0.62 g N-Methylpyrrolidone Ether Procedure. Dissolve biotinyl-N-e-aminocaproic acid in 20 ml Nmethylpyrrolidone, j2 Add the p-nitrophenol followed by the dicyclohexylcarbodiimide and stir gently overnight at room temperature. Filter off the dicyclohexylurea which forms. The product is precipitated by ether and collected on a sintered glass funnel. Rinse thoroughly with ether and dry in a vacuum desiccator.
Preparation of Biocytin Hydrazide (BCHZ) Reagents Biocytin, 1 g Hydrazine hydrate (Merck) Thionyl chloride (Fluka) Methanol Ether Dimethylformamide Distilled water Porapak Type Q column (Waters Associates Inc., Milford, MA), 10× I c m Procedure. Thionyl chloride (2.5 ml) is added to a solution of methanol (25 ml) cooled in an ice water-acetone bath. Biocytin (1 g) is then added, and the mixture is allowed to react overnight with constant stirring. The solvent is reduced to a minimal volume, and ether is added. The precipitate is filtered, washed with ether, and dried over NaOH under reduced pressure. TLC of the product (biocytin methyl ester) on silica gel gives one spot [Rf 0.48, butanol : acetic acid : water (4 : 1 : 1)] with no visible
[13]
BIOTIN-CONTAININGREAGENTS
131
contaminants using dimethylaminocinnamaldehyde 7 and ninhydrin sprays. The methyl ester of biocytin (1 g) is dissolved in 20 ml of methanol, and hydrazine hydrate (1 ml) is added. After 48 hr at room temperature, the solvent is concentrated to dryness; drying is continued further in a desiccator under reduced pressure in the presence of H 2 8 0 4 until the odor of hydrazine can no longer be detected. The product is dissolved in water (10 ml) and passed through a column containing Porapak Type Q. The column is eluted with water, and 10-ml fractions are collected.13 Fractions containing biocytin hydrazide are detected on TLC ( R f 0. I 1, using methanol as the solvent system) by dimethylaminocinnamaldehyde 7 and ninhydrin sprays. TM The fractions containing the product are pooled, concentrated to dryness, dissolved in a minimal amount of methanol, and precipitated with ether. Biocytin hydrazide can be crystallized from hot ethanol. On reaction with acetone, three reaction products are obtained (TLC, using methanol as solvent) with R f values of 0.24, 0.55, and 0.65, representing the anticipated reaction products. We have also prepared the same compound from t-Boc biocytin 15and tert-butyl carbazate in the presence of dicyclohexylcarbodiimide in a dimethylformamide solution. The protecting groups are removed with 4 N HCI in dioxane.
Preparation of 3-(N-Maleimidopropionyl)biocytin (MPB)16 Reagents Biocytin hydrochloride, I g in 10 ml of 0.5 M sodium bicarbonate 3-(N-Maleimidopropionyl)-N-hydroxysuccinimide ester (Calbiochem, San Diego, CA), 0.7 g Dimethylformamide 1 N HC1 Ether Procedure. 3-(N-Maleimidopropionyl)-N-hydroxysuccinimide ester is dissolved in dimethylformamide (10 ml) and added dropwise to the biocy13 The first several fractions usually contain residual hydrazine hydrochloride. It is essential to remove all of the latter since hydrazine reacts well with aldehydes and, as an impurity, would reduce the final efficiency of the hydrazide reagent. t4 To hasten elution of the hydrazide, the column is eluted with 80% aqueous methanol following the hydrazine peak. The fractions are then eluted as a sharp peak. ~5E. Bayer and M. Wilchek, this series, Vol. 34 [20]. 16 MPB and similar maleimido analogs of biocytin are now available from Sigma and Calbiochem.
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GENERALMETHODOLOGY
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tin solution. The reaction is carried out for 2 hr, after which the solution is acidified with 1 N HCI. The product is allowed to crystallize at 4°. The crystals are washed with cold 0.1 N HC1 and then dried with ether. Yield, 0.8 g (60%). MPB appears on TLC as a single spot, Rf 0.3, chloroform : methanol (1 : 1). The product can be detected by dimethylaminocinnamaldehyde,7 iodine vapors, or by burning the TLC plate. In order to test the reactivity of the spot to sulfhydryls, a 5-fold molar excess of dithiothreitol is interacted with MPB. The free sulfhydryl-containing derivative, MPB-SH, is formed. Unreacted dithiothreitol (Rf 0.9) is separated from the sulfhydryl adduct (Rf 0.35) using the above TLC system. Both dithiothreitol and MPB-SH are detected using 5,5'-dithiobis(2-nitrobenzoic acid) spray (0.1% in ethanol :0.1% NaHCO3, 1:1, v/v). Only the biotin-containing derivative is visible using dimethylaminocinnamaldehyde spray. 7
Preparation of Other Maleimidobiocytin Derivatives Other maleimido derivatives of biocytin can be prepared similarly, using commercially available maleimido aliphatic or aromatic N-hydroxysuccinimide esters. The aromatic derivatives are less soluble and hence less effective. 4-(N-Maleimidobutyryl)-N-hydroxysuccinimide ester and 6-(N-maleimidocaproyl)-N-hydroxysuccinimide ester are commercially available (Calbiochem, Sigma). The biocytin derivatives prepared from these compounds behave similarly to MPB.
Preparation of p-Aminobenzoylbiocytin N-t-Boc-p-aminobenzoic Acid Reagents p-Aminobenzoic acid (Fluka), 13.7 g BOC-ON (Fluka), 20 g 1 N NaOH Dioxane Petroleum ether Distilled water 10% Citric acid Procedure. p-Aminobenzoic acid is dissolved in 1 N NaOH (120 ml), and BOC-ON (dissolved in 50 ml dioxane) is added. The reaction mixture is stirred vigorously overnight. Water is added, and the solution is extracted with petroleum ether to remove the dioxane. The aqueous layer is acidified with citric acid, and the precipitate is collected, washed with
[13]
BIOTIN-CONTAININGREAGENTS
133
water, and air dried. Yield, 15 g (65%); mp 185°. TLC on silica plates, ether:petroleum ether (17: 3), yields a single spot (Rf 0.44) which could be distinguished from the starting material (Rf 0.40) by its insensitivity to ninhydrin staining.
t-Boc-p-aminobenzoyl-N-hydroxysuccinimide Ester Reagents N-t-Boc-p-aminobenzoic acid (6 g) N-Hydroxysuccinimide (Sigma), 3 g Dicyclohexylcarbodiimide (Fluka), 5.5 g Dioxane Ether Procedure. To the N-hydroxysuccinimide in dioxane (100 ml), solid N-t-Boc-p-aminobenzoic acid is added. The dicyclohexylcarbodiimide (dissolved in 20 ml dioxane) is added, and the solution is stirred for I hr. The filtrate is concentrated to minimal volume, and the product is crystallized from ether. Yield, 6.5 g (78%); mp 165°. TLC, ether:petroleum ether (19: 1), gives a single spot with gf 0.47 compared to 0.57 for the starting material.
t-Boc-p-aminobenzoylbiocytin Reagents Biocytin hydrochloride, 1 g, dissolved in 11 ml of 0.5 N NaOH t-Boc-p-aminobenzoyl-N-hydroxysuccinimide ester, 0.85 g Dimethylformamide Ether Petroleum ether Distilled water 10% Citric acid Procedure. t-Boc-p-aminobenzoyl-N-hydroxysuccinimide ester is dissolved in 10 ml of dimethylformamide, and biocytin is added. Additional dimethylformamide is added if a precipitate is formed, and the reaction is carried out overnight at room temperature. Water is then added, and the reaction mixture is extracted successively with ether and petroleum ether in order to remove the dimethylformamide. The solution is acidified with citric acid, and the precipitate is collected by filtration. The precipitate is washed with ether to remove traces of the reactants. Yield, 1.2 g (85%); mp 171°. TLC, butanol : acetic acid : water (4 : 1 : 1), gives a single spot with an Rf of 0.6 compared to 0.14 for biocytin.
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GENERALMETHODOLOGY
[13]
p-Aminobenzoylbiocytin (TFA salt) Reagents t-Boc-p-aminobenzoylbiocytin, 1.2 g Trifluoroacetic acid (TFA) Dichloromethane Ether Procedure. t-Boc-p-Aminobenzoylbiocytin is dissolved in 20 ml of a 1 : 1 mixture of TFA and dichloromethane and left at room temperature for 30 min. Ether is added, and the precipitate is filtered and washed with ether until dry. Yield, 1.05 g (88%). TLC, butanol:acetic acid:water (4 : 1 : 1), gives a single spot ( R f 0.4). On reaction with NaNO2, the product reacts with tyrosine.
p-Diazobenzoylbiocytin (DBB) 17 Reagents p-Aminobenzoylbiocytin, 2 mg 2 N HCi NaNOz, 7.7 mg/ml (ice-cold aqueous solution) 1 N NaOH 0.1 M Borate buffer, pH 8.4 Procedure. p-Aminobenzoylbiocytin is dissolved in ice-cold 2 N HCI (40.7/zl). An equivalent volume of NaNO2 is added, and the reaction is allowed to continue for 5 min at 4°. The reaction is terminated on addition of 1 N NaOH (35/zl). The DBB solution is brought to the desired concentration by dilution with borate buffer, and the solution is used immediately. Biotin-Containing Cross-Linking Reagents A new class of probes includes a series of trifunctional reagents in the general form B X--C--Y
where B includes the bicyclic ring system of biotin, C is a bifurcated spacer arm, and X and Y are reactive functional groups. The latter groupspecific functions can either be homofunctional (e.g., two N-hydroxysuccinimide groups) or heterofunctional (e.g., a maleimide and hydrazide 17 Diazobenzoyl derivatives are chemically unstable and cannot be stored for long periods of time. The preparation of DBB from the aminobenzoyl derivative is therefore performed immediately prior to use. The precursor is now available commercially from Calbiochem.
[13]
BIOTIN-CONTAININGREAGENTS
135
group). The spacer may be extended in any direction by addition of o~amino acids, in order to facilitate vectorial interaction with target molecules or with the avidin-containing probe. The spacer either can be uncleavable or can theoretically be cleavable in one, two, or three directions; the number and site(s) of cleavage on a biotinylated crosslinking reagent would be determined by the ultimate goal of the researcher, i.e., whether cleawtge of the biotin moiety is required to facilitate isolation (release from an avidin column) or whether cleavage of cross-linked macromolecules is desired for identification purposes (e.g., on two-dimensional SDS-PAGE). Suitable spacers may include mono-, di-, oligo-, and polysaccharides and their substituted derivatives. Alternatively, certain synthetic and naturally occurring amino acids (e.g., lysine, ornithine, aspartic and glutamic acids), dipeptides, and oligopeptides are also suitable as bifurcated spacers. Tartryl (periodate-cleavable) and disulfide (thiol-cleavable) derivatives can be used as cleavable extender arms. A wide variety of such reagents can be designed, some of which are presented in Fig. 3. The bifurcated biotin-containing reagents can be used for preparing conjugates of two different protein species, such as a monoclonal antibody and an enzyme. Alternatively, these reagents can be used for combined cross-linkage and biotinylation of molecular components of a complex biological system, such as the cell membrane. The presence of the biotin moiety allows subsequent isolation, identification, or targeting of the cross-linked molecules of interest. Some examples in the use of these reagents are presented later in this volume. 2
Preparation of 3-(N-Maleimidopropionyl)biocytin N-Hydroxysuccinimide Ester Reagents MPB, 523 mg N-Hydroxysuccinimide,6 125 mg Dicyclohexylcarbodiimide, 225 mg Dimethylformamide Ether Procedure. 3-(N-Maleimidopropionyl)biocytin is dissolved in 5 ml of dimethylformamide, and the N-hydroxysuccinimide is added. The solution is cooled to 4°, and dicyclohexylcarbodiimide is added. The reaction mixture is maintained for 24 hr at 4°. The dicyciohexylurea precipitate is filtered and discarded, and the product is precipitated directly from the filtrate with ether. The precipitate is collected, washed with ether, and the amount of N-hydroxysuccinimide ester is determined spectrophotometrically (/3261 10,000 M 1 cm 1) after hydrolysis with 0.5 M NH4OH.
136
GENERAL METHODOLOGY
0 II / c\ HN NH ~~
[13]
0 II /c\ HN NH
(CH2)4_c= 0 I NH I (CH2)4
~'~(CHz)4_C= 0 I NH X I -OC-(CH 2)2-CH-C0- y
I
X -NH-CH-CO- y
Functional groups
for X and
y
0
Spacers Noncleavable
-0-1~
HOOC-(CHz)n- COOH HzN-(CH2)n-NHz HzN-(CH2)n- COOH
-0"-~'-
NOz
HzN- (CHz)4-CH-COOH
o
%
-C- (CHz)n-N~ I
Cleavable
o
HOOC-[CHz)2-S-S- (CHz)2-COOH
-NHNH z 0 II /-~-~ + -C ~-4~.~N z 0 /-~'~ + -CII~-'~,..~--N~
or
or
HzN-
- N H - - ~ - - N;
-NH-~
N;
{CH2]2 - $ - $ - (C H2)2-
NH2
HOOC-CH- CHz- S-S-CH2-CH-COOH HO OH I I HOOC-CH-CH-COOH OH OH I I HOOC-(CHz)n-HNOC-CH-CH-CONH-(CHz)n-COOH
FIG. 3. Examples of biotin-containing cross-linking reagents. The examples shown are based either on biocytin, which provides both an amine and a carboxyl group for further derivatization with reactive groups X and Y, or on biotinyl glutamate, which provides two free carboxyl groups for the same purpose. The reactive functional groups X and Y can either be identical (in the case of homobifunctional reagents) or different (heterobifunctional reagents). In some cases, a given functional group is incompatible with another (e.g., a hydrazide derivative on one side and a p-nitrophenyl or N-hydroxysuccinimide group on the other), since the reagent would polymerize. The reactive groups and/or the biotin moiety can be extended further from the point of bifurcation by the incorporation of a suitable spacer group. If the latter is a cystinyl or tartryl derivative, the resultant reagent would be cleavable at the corresponding position.
[13]
BIOTIN-CONTAININGREAGENTS
137
Preparation of 3-(N-Maleimidopropionyl)biocytin Hydrazide Reagents 3-(N-Maleimidopropionyl)biocytin N-hydroxysuccinimide ester, 125 mg in 1 ml of dimethylformamide Hydrazine hydrate, 10/.d in 1 ml of 0.5 M sodium bicarbonate Methanol Ether Procedure. The solution of 3-(N-maleimidopropionyl)biocytin Nhydroxysuccinimide is added dropwise to the hydrazine hydrate solution. The reaction is carried out for 3 hr at room temperature. The solution is concentrated to dryness, dissolved in a minimal amount of methanol, and the product is precipitated with ether. Purity is determined by TLC, and the product is identified by its reaction with acetone (and consequent change in Rf value). Other hydrazide derivatives bearing long spacer arms are prepared by substituting 3 to 5 molar equivalents of the respective dihydrazide (e.g., 100-200 mg adipic acid dihydrazide). These derivatives can also be prepared directly from 3-(N-maleimidopropionyl)biocytin using either hydrazine or a desired dihydrazide derivative in the presence of dicyclohexylcarbodiimide.
Biotinyl-L-glutamic Acid Reagents BNHS, 3.4 g in 30 ml of dimethylformamide L-Glutamic acid, 1.5 g in 30 ml of 0.5 M sodium bicarbonate l N HCI Distilled water Procedure. The BNHS and glutamic acid solutions are mixed, and the reaction is carried out for 2 hr at room temperature. The solution is acidified with HC]; the resultant precipitate is washed with cold water and dried in vacuo. Other aminodicarboxylic acids, peptides, etc., are prepared by substituting equimolar amounts for glutamic acid.
Biotinyl-L-glutamic Acid Dihydrazide Reagents Biotinyl-L-glutamic acid, 1 g in 10 ml of methanol Thionyl chloride, 1 ml Methanol Sodium hydroxide
138
GENERALMETHODOLOGY
[14]
Hydrazine hydrate, l ml 2-Propanol Procedure. The thionyl chloride and the methanolic solution of biotinyl-L-glutamic acid are cooled separately in an acetone-dry ice bath before mixing. The reaction is left at room temperature for 24 hr. The methanol is removed under vacuum; an equivalent volume of methanol is added and removed again by evaporation, and this step is repeated twice again. The residue is dried in a desiccator over sodium hydroxide (to remove traces of HCI). The residue is again dissolved in methanol, hydrazine hydrate is added, and the reaction is left at room temperature for 24 hr. A portion of the biotinylglutamic acid dihydrazide precipitates out and is filtered. The remainder is obtained by evaporation of the methanol and crystallization from 2-propanol.
[14] P r o t e i n B i o t i n y l a t i o n
By EDWARD A. BAYER and MEIR WILCHEK An essential prerequisite in the successful application of avidin-biotin technology is the incorporation of the biotin moiety into the experimental system. This may be accomplished by direct biotinylation of the target system using one of the many examples of group-specific biotin-containing reagents described elsewhere in this volume. J However, another alternative, one that contributes toward the virtually unlimited potential of the system, is the capacity to biotinylate biologically active molecules which maintain their recognition properties toward a given target molecule? -4 In this case, biotin may be introduced chemically via a variety of functional groups on the binder using the same group-specific reagents. In this chapter, we present some of the procedures by which we have incorporated biotin moieties into proteins. Although in earlier w o r k s 4,5 w e have professed that biotinylation of a macromolecule is relatively harmless to its activity and only minimally affects its physical and chemical properties, this is not necessarily true, and a given biotinylated binder should always be thoroughly characterized prior to use. We therefore M. 2 E. 3 E. 4 E. 5 M.
Wilchek and E. A. Bayer, this volume [131. A. Bayer, M. Wilchek, and E. Skutelsky, F E B S Lett. 68, 240 (1976). A. Bayer and M. Wilchek, Trends Biochem. Sci. 3, N257 (1978). A. Bayer and M. Wilchek, M e t h o d s Biochem. Anal. 26, 1 (1980). Wilchek and E. A. Bayer, l m m u n o l . Today 5, 39 (1984).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 184
Copyright .~; 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
