Hermanson G. Bioconjugate Techniques, Second Edition

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320 5. Heterobifunctional Crosslinkers

Figure 5.27 SAED may be used to transfer the ﬂ uorescent AMCA label from the ﬁ rst molecule modiﬁ ed with

the crosslinker to the second molecule crosslinked with it by reduction of its internal disulﬁ de bond. Thus,

unknown target molecules may be ﬂ uorescently tagged to follow them in vivo .

SAED, however, sulfo-SAMCA contains a short non-cleavable cross-bridge (12.8 Å) where the

active ester functionality is constructed directly off the carboxylate group of AMCA without

any other intervening spacer groups. Conjugated molecules will retain the ﬂ uorescent label,

thus providing detectability to the complexes formed ( Figure 5.28 ). However, since crosslinks

3. Amine-Reactive and Photoreactive Crosslinkers 321

Figure 5.28 Sulfo-SAMCA can be used to modify amine-containing molecules through its NHS ester end.

Subsequent exposure to UV light causes bond formation with nearby nucleophilic groups, such as amines. The

photosensitive phenyl azide group is created on the aromatic ring of an AMCA ﬂ uorophore. Before photoacti-

vation, the azide group makes the crosslinker nonﬂ uorescent. After photoactivation, however, the azide group is

either lost by N

generation or couples to a target molecule. Either way, the AMCA portion becomes ﬂ uorescent

to allow tracking of the conjugate. The photoreaction may occur through ring expansion to an intermediate

dehydroazepine or might happen through nitrene formation.

322 5. Heterobifunctional Crosslinkers

formed with this reagent are not cleavable, sulfo-SAMCA cannot function as a ﬂ uorescent label

transfer agent in the fashion of SAED.

3.11. p -Nitrophenyl Diazopyruvate

Diazopyruvates represent a unique class of photoreactive reagents that are not often used in

heterobifunctional crosslinker design. The p-nitrophenyl ester derivative of diazopyruvate provides

amine-reactive, acylating potential, while the photosensitive group can be activated with UV light

to generate reactive aldehydes. More speciﬁ cally, the diazo functionality can be photolyzed by

exposure to irradiation at 300 nm, forming a highly reactive carbene which can undergo a Wolff

rearrangement that produces a ketene amide intermediate. In the presence of a nucleophilic spe-

cies on a target molecule, the ketene can undergo an acylation reaction to form a stable malonic

acid derivative. The photolyzed product thus can couple to hydrazide- or amine-containing targets

to form covalent linkages ( Figure 5.29 ).

p-nitrophenyl diazopyruvate (Invitrogen) is relatively insoluble in water or aqueous buff-

ers, but may be pre-dissolved in DMF before adding an aliquot of the stock solution to an

aqueous reaction mixture. All solutions of the reagent should be carefully protected from

light to prevent premature photolysis. p-nitrophenyl diazopyruvate has an absorbance maxi-

mum at 390 nm with a molar extinction coefﬁ cient of about 19,000 M

 1

in methanol.

p-nitrophenyl diazopyruvate has been used in the photoreactive crosslinking of calmod-

ulin with adenylate cyclase from bovine brain (Harrison et al., 1989), to crosslink aldolase

(Goodfellow et al., 1989), as a bonding agent for tissue containing type-I collagen (Givens

et al., 2003) or to bond to corneal tissue (Timberlake et al., 2005), and in the photoreactive

coupling of DNA to paramagnetic beads (Penchovsky et al. , 2000).

3.12. PNP-DTP

PNP-DTP, p-nitrophenyl-2-diazo-3,3,3-triﬂ uoropropionate, is a photoreactive heterobifunc-

tional crosslinker that contains an amine-reactive group on one end and a photosensitive diazo

group on the other (Chowdhry et al., 1976) (Thermo Fisher). p-nitrophenyl esters react similarly

Figure 5.29 pNPDP reacts with amine-containing compounds by its p-nitrophenyl ester group to form amide

bonds. After photoactivation of the diazo derivative with UV light, a Wolff rearrangement occurs to a highly

reactive ketene intermediate. This group can couple to nucleophiles such as amines.

3. Amine-Reactive and Photoreactive Crosslinkers 323

324 5. Heterobifunctional Crosslinkers

to NHS esters (Chapter 4, Section 1 and Chapter 2, Section 1.4) but in this case, with p-nitrophenol

as the leaving group upon reaction with a nucleophile. Amine-containing target molecules such

as proteins can be modiﬁ ed with this reagent to form amide bond derivatives possessing pho-

toactivatable functionalities. The reagent is small enough to probe deep within the active centers

of receptor molecules and other sites of biomolecular interactions ( Figure 5.30 ).

PNP-DTP has been used to photoafﬁ nity label the thyroid hormone nuclear receptors in

intact cells by preparing a derivative of 3,5,3  -triiodo- L-thyronine with the crosslinker (Pascual

et al., 1982; Casanova et al., 1984). Effective photoreactive conjugation was found to occur

after irradiation with UV light at 254 or 310 nm.

4. Sulfhydryl-Reactive and Photoreactive Crosslinkers

The beneﬁ ts of nonselective photoreactive crosslinking can be merged with the directed cou-

pling ability of sulfhydryl-reactive functionalities to create heterobifunctional reagents pos-

sessing greater utility than the standard amine and photoreactive agents discussed previously.

Having a sulfhydryl-reactive group on one end of the crosslinker allows the initial conjugation

to take place at more discrete sites on proteins and other molecules before irradiation to effect

the ﬁ nal photosensitive reaction.

Figure 5.30 PNP-DTP can modify amine-containing molecules through its p-nitrophenyl ester group to form

amide bonds. Exposure of its photosensitive diazo group with UV light generates a highly reactive carbene that

can insert into active C  H or N  H bonds.

The following reagents contain a variety of sulfhydryl-reactive groups, including iodoacetyl

derivatives, maleimide compounds, and pyridyl disulﬁ de chemistries. The iodoacetyl and maleimide

functions form permanent thioether bonds with target molecules containing free sulfhydryls.

The pyridyl disulﬁ de derivative reacts with  SH groups to form reversible disulﬁ de linkages,

which can be cleaved with disulﬁ de reducing agents like DTT.

The photoreactive end of the following crosslinkers also varies from the traditional aryl

azide group to the newer benzophenone and ﬂ uorinated aryl azide derivatives. The ﬂ uorinated

phenyl azide functional groups photolyze to true nitrenes without the ring-expansion side reac-

tion characteristic of aryl azides. The result is that ﬂ uorinated aryl azides more effectively insert

into active carbon–hydrogen bonds, rather than potentially undergoing nucleophilic reactions

like phenyl azides. In addition, benzophenone groups generally have higher degrees of bond

formation with the intended target molecule compared to the yields obtained using traditional

phenyl azides, due to their ability to be repeatedly photolyzed without breakdown of the pre-

cursor to an inactive form.

The number of commercially available crosslinkers for sulfhydryl and photoreactive con-

jugations provides enough variety to design successful experiments in photolabeling, such as

studying active centers and macromolecular interactions.

4.1. ASIB

ASIB, 1-( p-azidosalicylamido)-4-(iodoacetamido)butane, is a heterobifunctional crosslinker con-

taining a sulfhydryl-reactive iodoacetyl group on one end and a photosensitive phenyl azide

group on the other end (Thermo Fisher). The phenyl azide ring is substituted with a ring-activat-

ing hydroxyl group which provides the ability to radioiodinate the compound before the con-

jugation reaction is performed. Since both the iodoacetyl and phenyl azide functionalities are

relatively stable in aqueous solutions, the steps involved in iodination and crosslinking do not

detrimentally affect the subsequent reactivity of the reagent. All operations should be done pro-

tected from light, however, to prevent premature photolysis before the desired crosslinking reac-

tion is initiated. The cross-bridge of the reagent provides an 18.8 Å spacer between crosslinked

molecules.

The reaction of ASIB with sulfhydryl-containing molecules can be done at mildly alkaline

pH with excellent speciﬁ city. Higher pH conditions may cause cross-reactivity with amines.

4. Sulfhydryl-Reactive and Photoreactive Crosslinkers 325

326 5. Heterobifunctional Crosslinkers

Photolyzing with UV light may result in immediate reaction of the nitrene intermediate with a tar-

get molecule within Van der Waals distance, or may result in ring expansion to the nucleophile-

reactive dehydroazepine. The ring-expanded product is reactive primarily with amine groups

(Figure 5.31 ).

4.2. APDP

APDP, N-[4-(p-azidosalicylamido)butyl]-3-(2-pyridyldithio) propionamide, is a radioiodinat-

able, heterobifunctional crosslinking agent that contains a sulfhydryl-reactive pyridyl disulﬁ de

group on one end and a photosensitive phenyl azide on the other end (Thermo Fisher).

Radioiodinatable crosslinkers eliminate the need to radiolabel one of the reacting proteins,

thus avoiding potential activity losses due to modiﬁ cation of important residues (Chapter 12,

Section 5). They also allow radiolabeling of unknown target molecules which interact with

the initially modiﬁ ed protein. APDP reacts with sulfhydryl-containing proteins and other mole-

cules to form a reversible disulﬁ de bond. If the crosslinker is radiolabeled prior to conjugation,

cleavage of the disulﬁ de group with DTT after crosslinking effectively transfers the iodinated

portion to the secondary, photocoupled protein. This radiolabel transfer process allows track-

ing of a speciﬁ c receptor or other interacting species after conjugation with its complementary

Figure 5.31 ASIB can react with sulfhydryl-containing molecules through its iodoacetate group to form

thioether linkages. Subsequent exposure to UV light causes a ring-expansion process to occur, creating a highly

reactive dehydroazepine intermediate that can couple to amine-containing molecules.

ligand ( Figure 5.32 ). APDP thus falls into the general category of label transfer reagents that

can be used to study protein interactions (Chapter 28).

The reactions of APDP are similar to that of the reported compound N -(4-azidophenyl)

thiophthalimide, a non-radioiodinatable crosslinker (Moreland et al., 1982). Both the phenyl

4. Sulfhydryl-Reactive and Photoreactive Crosslinkers 327

Figure 5.32 APDP can modify sulfhydryl-containing compounds through its pyridyl disulﬁ de group to form

disulﬁ de bonds. Its phenyl azide end then can be photolyzed with UV light to couple with nucleophiles via a

ring-expansion process. The disulﬁ de group of the crosslink can be selectively cleaved using DTT.

328 5. Heterobifunctional Crosslinkers

azide group and the pyridyl disulﬁ de portion are stable in aqueous environments prior to the

crosslinking reaction. The initial modiﬁ cation with a sulfhydryl-containing protein should be

done protected from light to preserve the activity of the photosensitive group. Avoid, also, in

the reaction medium disulﬁ de reducing agents that can react with the pyridyl disulﬁ de group as

well as inactivate the phenyl azide portion.

The cross-bridge of APDP provides a long, 21.02 Å spacer that is able to reach distant

points between two interacting molecules. Cleavage of the crosslink with a disulﬁ de reducing

agent regenerates the original sulfhydryl-modiﬁ ed protein without leaving any other chemi-

cal groups behind. The remainder of the crosslinker stays attached to the second, interacting

protein.

APDP is soluble in DMSO and DMF, but almost insoluble in acetone or water. Stock solu-

tions may be prepared in DMSO or DMF and a small aliquot added to an aqueous reaction

mixture. Do not exceed 10 percent organic solvent in the buffered reaction. Both functionalities

of APDP will react in a variety of salt conditions and pH values. For reaction with a sulfhydryl-

containing protein, a buffer at physiological pH containing a chelating agent to protect the free

sulfhydryl groups from metal-catalyzed oxidation is recommended (i.e., 0.01–0.1 M sodium

phosphate, 0.15 M NaCl, pH 7.2, containing 10 mM EDTA).

Iodination of the crosslinker may be done according to the procedures discussed in Chapter

12, Section 5, or performed similar to that described for SASD (Section 3.2, this chapter).

4.3. Benzophenone-4-iodoacetamide

A photoreactive group consisting of a benzophenone residue photolyzes upon exposure to UV

light to give a highly reactive triplet-state ketone intermediate (Walling and Gibian, 1965).

Similar to the reactive nitrene of photolyzed phenyl azides, the energized electron of an acti-

vated benzophenone can insert in active hydrogen–carbon bonds and other reactive groups to

give covalent linkages with target molecules. Unlike phenyl azides, however, the decomposi-

tion or decay of the photoactivated species does not yield an inactive compound. Instead,

benzophenones that have become deactivated without forming a covalent bond can be once

again photolyzed to an active state. The results of this multiple-activation characteristic are

more than one chance to form a crosslink with the intended target and much higher yields of

photo-crosslinking.

The heterobifunctional crosslinker benzophenone-4-iodoacetamide is a photoreactive reagent

containing a sulfhydryl-reactive iodoacetyl derivative at one end and a benzophenone group on

the other end (Hall and Yalpani, 1980; Tao et al., 1984; Lu and Wong, 1989) (Invitrogen).

The iodoacetyl group has similar reactivity to the same group on the heterobifunctional reagent

SIAB (Section 1.5, this chapter). Under alkaline conditions (pH 8–9), the iodoacetyl reaction is

highly speciﬁ c for sulfhydryl residues in proteins and other molecules, forming stable thioether

linkages. The initial modiﬁ cation reaction of sulfhydryl-containing compounds should be done

protected from light to avoid premature photolysis of the benzophenone functionality. Also,

avoid thiol-containing reducing agents in the sample, as these will react with the iodoacetyl

group. After puriﬁ cation of the benzophenone-modiﬁ ed protein from excess reagent (by dialy-

sis or gel ﬁ ltration), it is mixed with a sample containing a second target molecule (e.g., a cell

lysate) to allow an interaction to take place, and then photolyzed with UV light to effect the

ﬁ nal crosslink ( Figure 5.33 ). Since repeated photolysis of the benzophenone species is possible,

the yield of such conjugation reactions can be signiﬁ cantly higher than using other photoreac-

tive groups. One report indicated that crosslinking with chymotrypsin approached 100 percent

efﬁ ciency (Campbell and Gioannini, 1979).

Benzophenone-4-iodoacetamide is water-insoluble and should be pre-dissolved in DMF or

another organic solvent prior to adding an aliquot to an aqueous reaction mixture. Stock solu-

tions may be prepared and stored successfully if protected from light.

Benzophenone-4-iodoacetamide has been used to study the 100-KDa U5 snRNP protein

(hPrp28p) and its interactions (Ismaili et al. , 2001).

4. Sulfhydryl-Reactive and Photoreactive Crosslinkers 329

Figure 5.33 Benzophenone-4-iodoacetamide reacts with sulfhydryl-containing compounds to give thioether

linkages. Subsequent photoactivation of the benzophenone residue gives a highly reactive triplet-state ketone

intermediate. The energized electron can insert in active C  H or N  H bonds to give covalent crosslinks.