Hermanson G. Bioconjugate Techniques, Second Edition

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670 17. Chemoselective Ligation: Bioorthogonal Reagents

Figure 17.3

Maleimide-modiﬁ ed glass slides (1) can be derivatized using two chemoselective ligation reac-

tions to create biotin modiﬁ cations. In the ﬁ rst step, alkyne–PEG

–cyclopentadiene linkers (2) are added to the

maleimide groups using a Diels–Alder reaction. In the second reaction, an azido-PEG

–biotin compound (3) is

reacted with the terminal alkyne on the slide using click chemistry to result in another cycloaddition product, a

triazole ring.

successful bioconjugation reactions done with oxidized glycoproteins on cell surfaces or in

lysates (Bayer et al ., 1987a, b, 1990; Bayer and Wilchek, 1990).

The hydrazine–aldehyde reaction has been used intracellularly to deliver non-toxic drug

components, which when linked to form a hydrazone bond in situ, become cytotoxic (Rideout,

1986, 1994; Rideout et al., 1990). This same approach has been used to generate enzyme

inhibitors in vivo, wherein the hydrazine and aldehyde precursors are not active, but when cou-

pled together within cells to form a hydrazone linkage, become active site binders (Rotenberg

et al ., 1991).

Ketone containing bio-monomer analogs have been used successfully to incorporate these

functionalities into biopolymers, such as proteins and carbohydrates (glycans). For example, a

phenylalanine analog containing a para-substituted aryl ketone group is able to be transformed

into an aminoacyl t-RNA and then introduced with sequence speciﬁ city into a protein by cel-

lular ribosomal machinery (Datta et al., 2002). In addition, the post-translational glycosylation

process within cells is tolerant of certain sugar analogs, and ketone derivatives of monosac-

charides have been used to incorporate these functions into glycans. In particular, growing cells

in the presence of N -levulinoyl- D-mannosamine (ManLev; Figure 17.4 ) was found to result in

the incorporation of this ketone sugar into cell-surface carbohydrates through enzymatic trans-

formation into a keto sialic acid (Mahal et al., 1997; Lemieux and Bertozzi, 1999). Since sialic

acid residues are terminal sugars on most mammalian cell-surface glycoproteins, the addition of

pendent ketone groups allows for speciﬁ c targeting of these glycans with hydrazine (hydrazide/

aminoxy) based reagents.

There are many bioconjugate reagents and probes available with aldehyde or hydrazide func-

tional groups that may be used for hydrazone bond formation. However, the best choices for

making this reaction as chemoselective and bioorthogonal as possible are aromatic aldehyde and

aromatic hydrazine derivatives due to their extremely low reactivity with biological functional

groups, minimal nonspeciﬁ c interactions with biomolecules (charge or Schiff base formation),

and their highly efﬁ cient hydrazone bond formation in complex aqueous solutions (Solulink,

Thermo Fisher). Reagents of this type typically contain reactive groups consisting of either an

aryl aldehyde (benzaldehyde) or a 6-hydrazinium nicotinate (Schwartz et al., 1993, 1995).

Figure 17.4 Ketone derivatives of phenylalanine and mannose can be fed to cells to incorporate the monomers

into proteins and glycans. The resultant modiﬁ cations can be probed using hydrazide-containing reagents.

2. Hydrazine–Aldehyde Reagent Pairs 671

672 17. Chemoselective Ligation: Bioorthogonal Reagents

These aldehyde and hydrazine functionalities can be associated with virtually any other

reactive group or tag, such as heterobifunctional crosslinkers, ﬂ uorescent labels, or biotin

compounds. Two reagents frequently used to chemically introduce the aldehyde and hydra-

zine groups include SANH (succinimidyl 4-hydrazinonicotinate acetone hydrazone) and SFB

(succinimidyl 4-formylbenzoate) ( Figure 17.5 ). Long-chain analogs of these two compounds

also exist, which contain either a 6-aminocaproyl spacer arm in the cross-bridge or a short

PEG spacer to promote increased water solubility. The SFB and SANH-NHS ester compounds

are amine-reactive and form amide bonds with the molecules modiﬁ ed by them ( Figure 17.6 ).

Other reactive groups or tags also are available having the benzaldehyde or hydrazinium nico-

tinate groups, including maleimide (thiol-reactive), biotin, and various ﬂ uorescent probes

(Solulink).

The hydrazinium nicotinate group on these reagents commonly is protected against reaction

with the active ester by the addition of acetone to form the acetone hydrazone derivative. This

hydrazone protective group is readily reversible at neutral or mildly acidic pH and will imme-

diately exchange with a benzaldehyde on the corresponding chemoselective partner to form a

stable hydrazone linkage.

Figure 17.5 Structures of aldehyde-containing and hydrazine-containing heterobifunctional crosslinkers that

can be used in chemoselective hydrazone conjugation procedures.

Proteins, other molecules, or surfaces that are activated to contain either the benzaldehyde

functionality or the hydrazinium nicotinate group can be quantiﬁ ed as to the number of these

groups present using certain chromophoric reactants. For instance, 2-hydrazinopyridine can

be reacted with a benzaldehyde-containing molecule to give a UV absorbing derivative with



max

 350 nm and an extinction coefﬁ cient of 18,000 /cm

1

. Conversely, the hydrazinium

nicotinate-activated molecules can be reacted with p-nitrobenzaldehyde to create another UV

detectable derivative having 

max

 390 nm with an extinction coefﬁ cient of 24,000 cm

1

The aromatic nature of the SFB and SANH reactants create a hydrazone structure with

unique absorptivity for monitoring the course of the ligation process. The aromatic hydra-

zone bond has a maximal absorbance at 354 nm and a molar extinction coefﬁ cient equal to

Figure 17.6 The reaction of SANH with amine-containing proteins or other molecules results in amide bond

modiﬁ cations containing terminal hydrazine groups. The reaction of SFB with amine-containing proteins or

other molecules results in amide bond modiﬁ cations containing terminal aldehyde groups. Subsequently, the

two modiﬁ ed molecules can be reacted together to create a conjugate via hydrazone bond formation.

2. Hydrazine–Aldehyde Reagent Pairs 673

674 17. Chemoselective Ligation: Bioorthogonal Reagents

29,000/M. If the conjugation reaction is done with sufﬁ cient amounts of benzaldehyde and

hydrazinium nicotinate reactants, then the yield of the reaction can be directly measured by

monitoring the change in absorbance at this wavelength over time.

The bis-aryl hydrazone bond formed by this reaction is stable in aqueous solution over a broad

pH range (pH 2–11) and under elevated temperature conditions (up to 94 °C) (Solulink web site).

Kozlov et al. (2004) used SANH and SFB to create DNA-antibody conjugates for highly sen-

sitive detection in immunoassays using PCR ampliﬁ cation with ﬂ uorescently labeled primers.

They also investigated hydrazone bond formation and its stability in aqueous solution using

3 and 5  labeled DNA pairs, wherein one oligonucleotide contained a benzaldehyde group and

the other contained the hydrazinium nicotinate group. It was found that the most efﬁ cient pH

for the creation of the hydrazone bond was pH 5.0–7.0, with an optimum at about pH 6 and

yields dropping off at lower or higher pH. In addition, once the hydrazone bond was formed

and the conjugate puriﬁ ed, it was stable in PBS buffer, pH 7.2, with or without 10 mg/ml BSA

for 12 weeks at 4 °C. The hydrazone linkage also was found to be stable at pH values between

2.3 and 11.3 without hydrolytic breakdown.

When reactions between the benzaldehyde and hydrazinium nicotinate groups are done in rela-

tively dilute solution, for example using a modiﬁ ed antibody molecule at 1 mg/ml ( 6 M) con-

taining one of the two reactants, then to obtain maximal yield of the hydrazone conjugate, the

second component should be added in sufﬁ cient excess to drive the reaction to completion. Kozlov

et al. (2004) found that at least an 8-fold molar excess or above of the second reactant over the

ﬁ rst reactant is necessary to obtain a yield of about 80 percent hydrazone bond formation.

The practical use of chemoselective ligation reactions for bioconjugation purposes involves

the attachment of one of the reactants to a ﬁ rst biomolecule using a standard coupling chem-

istry (e.g., NHS ester) to an available functional group, while the second reactant is coupled

to a second molecule or a surface, depending on the ﬁ nal conjugate desired. In this regard, the

following protocols for modiﬁ cation of oligonucleotides or proteins can be done with either

the benzaldehyde component or the hydrazine component and the opposite reactant is used to

modify another molecule or surface that will be coupled in the ﬁ nal conjugation step.

Protocol for Modiﬁ cation of Amine-Oligo with SANH or SFB

1. Dissolve a 5  -amine modiﬁ ed oligonucleotide at a concentration of 0.5 mM in 0.1 M

sodium phosphate, 0.15 M NaCl, pH 7.4.

2. Dissolve SANH (or SFB) in DMF at a concentration of 100 mM. Note: both reagents are

soluble in this solvent to about 50 mg/ml concentration.

3. Add a quantity of SANH or SFB solution to the oligo to provide a 40-fold molar excess

of crosslinker over the amount of amine-oligo present. Mix well.

4. React at room temperature for 2 hours to overnight with gentle mixing.

5. Purify the modiﬁ ed oligo from unreacted crosslinker and reaction byproducts using a

micro-spin concentrator with a membrane having a molecular weight cutoff able to

retain the size of the DNA being modiﬁ ed.

A similar method can be used to modify proteins, such as antibodies, to contain either benz-

aldehyde or hydrazinium nicotinate groups for subsequent conjugation with another molecule

modiﬁ ed by the opposite functionality. The following protocol is based on the methods of Thermo

Fisher, Solulink, and Kozlov et al. (2004).

Protocol for Modiﬁ cation of Protein or Antibody with SANH or SFB

1. Dissolve the protein or antibody to be conjugated in 0.1 M sodium phosphate, 0.15 M

NaCl, pH 7.4. The antibody solution should be as concentrated as possible, given the

amount of antibody available for modiﬁ cation. This general protocol will work for

protein or antibody concentrations ranging from about 0.5 mg/ml to 10 mg/ml, but

an increase in the molar excess of either SANH or SFB may have to be done at lower

antibody concentrations to provide the same modiﬁ cation yields obtained at higher

concentrations.

2. Dissolve SANH or SFB in DMF at a concentration of 2 mg/100  l DMF.

3. Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solu-

tion to obtain the desired molar excess of reagent over the antibody. Typically, antibody

modiﬁ cation procedures are done with 10- to 20-fold molar excess, but for dilute anti-

body concentrations, this may have to be doubled, depending on how many hydrazine or

aldehyde groups are desired to be introduced on the modiﬁ ed antibody.

4. React for 2–3 hours at room temperature with gentle mixing.

5. Purify the modiﬁ ed antibody by use of size exclusion chromatography or dialysis, using a

molecular weight exclusion limit of 5,000–10,000 Daltons. The modiﬁ ed antibody may

be stored at 4°C or frozen until used to make a conjugate. The protected hydrazine on

SANH is stable for several months and the SFB benzaldehyde group is stable indeﬁ nitely.

For longer storage, the modiﬁ ed antibody can be lyophilized.

Once a molecule is modiﬁ ed with a hydrazine reagent and another molecule is modiﬁ ed with

the benzaldehyde compound, they may be combined to form the ﬁ nal conjugate, which will

result in a hydrazone linkage between the two molecules. In addition, chemoselective ligation

using aldehyde/hydrazine reactions may be done to immobilize biomolecules. In this regard,

one modiﬁ ed component may be a surface and the other one an antibody, protein, or oligonu-

cleotide destined for immobilization onto the surface.

Conjugation Using the Aldehyde/Hydrazine Reaction

1. Separately dissolve the SFB-modiﬁ ed molecule and the SANH-modiﬁ ed molecule in cit-

rate buffer (100 mM sodium citrate, 150 mM NaCl, pH 6.0) at concentrations of at least

1 mg/ml. Note that pH 6 is an optimal pH for the formation of the hydrazone bond, but

pH values slightly lower (to pH 4.7) or higher (to pH 7.4) also may work, but they will

result in lower reaction rates and lower yields.

2. Mix a portion of the SFB-modiﬁ ed molecule with the SANH-modiﬁ ed molecule to obtain

a molar ratio that will give the desired conjugate properties. The optimal ratio of reactants

may have to be determined experimentally by performing a series of conjugations using

different molar ratios and testing the performance of the ﬁ nal conjugate in the intended

application.

3. React for at least 2 hours at room temperature, longer if pH values other than pH 6 are

used.

4. To purify the conjugate from reactants that did get incorporated into the conjugate, size

exclusion chromatography may be used with resins having a molecular exclusion limit

able to accommodate both the labeled molecules and the ﬁ nal conjugate.

2. Hydrazine–Aldehyde Reagent Pairs 675

676 17. Chemoselective Ligation: Bioorthogonal Reagents

3. Boronic Acid–Salicylhydroxamate Reagent Pairs

Phenylboronic acid (PBA) groups can interact with a variety of polar constituents on adjacent

or nearby carbon atoms to result in a complex consisting of a 5- or 6-member heterocyclic ring.

This process has been used for the afﬁ nity chromatographic puriﬁ cation of carbohydrates, gly-

coproteins, RNA, AMP (from cAMP), glycated proteins (such as glycated hemoglobin formed

in diabetes; Klenk et al., 1982), and a range of small molecules containing 1,2- or 1,3-diols,

1,2- or 1,3-hydroxy acids, 1,2- or 1,3-hydroxylamines, 1,2- or 1,3-hydroxyamide, 1,2- or

1,3-hydroxyoxime, as well as various sugars containing these species (Weith et al., 1970;

Rosenberg and Gilham, 1971; Rosenberg et al., 1972; Pace and Pace, 1980; Singhal et al .,

1980). For a review on the use of PBA in afﬁ nity separations, see Scouten (1983). In addition,

bioconjugate labeling reagents containing a PBA group also have been used as probes of these

species in biological molecules, including ﬂ uorescent reagents for targeting glycans on cell sur-

faces (Burnett et al ., 1980; O ’Shannessy and Quarles, 1987).

The interaction of PBA derivatives with molecular species having the above functional

groups occurs optimally in the pH range of 8–9, but it is typically reversible at acid pH or in

the presence of a high concentration of competing ligand. However, the heterocyclic boronic

acid complex is relatively stable under optimal conditions of formation.

Stolowitz (1997) exploited this interaction potential in the design of a new chemoselective

bioconjugation reagent pair consisting of a PBA group on one reagent and a salicylhydroxamic

acid (SHA) group on a second reagent. Each reactant of the pair can be used to modify biomole-

cules, surfaces, or other compounds for subsequent conjugation or immobilization through spe-

ciﬁ c PBA–SHA ring formation (Springer et al., 2002). The major product of this reaction forms

a 6-membered ring structure consisting of the PBA ’s boron atom along with one of its oxygens

coordinated with the SHA ’s hydroxyl oxygen and hydroxamate nitrogen atoms ( Figure 17.7 ).

It also is possible that a 5-membered ring structure can form from interaction of the PBA boron

with the hydroxamate hydroxyl and carbonyl oxygens on SHA, but this is a minor product as

proven by NMR (Stolowitz et al., 2001).

A signiﬁ cant advancement in the PBA reagent was to add another boronic acid group to

the phenyl ring and thus allow two cycloaddition products to form from a single complex-

ation. The phenyldiboronic acid (PDBA) group effectively increases the afﬁ nity constant

of the interaction if reacting with SHA-modiﬁ ed molecules or surfaces that have more than

one near-neighbor SHA group available. The resultant formation of two 6-membered rings

per conjugation reaction assures that the bond won ’t hydrolyze even under high or low pH

conditions. This is particularly useful for using the reaction to couple proteins or other mol-

ecules to surfaces for arrays, because multiple linkages maintain stability of the immobilized

molecule without the possibility for leaching off. A single SHA–PBA bond reportedly has an

afﬁ nity constant in the range of 10

1

, which is relatively weak for afﬁ nity binding proper-

ties. Two such bonds, however effectively raise the avidity to an observed afﬁ nity constant of

10

1

(Lonza product information). Note that multiple single PBA groups also can com-

bine with multiple SHA groups when conjugating proteins together or proteins to surfaces and

thus increase the effective avidity of the resultant bonding interaction. Springer et al. (2003)

describes the use of SHA membranes in this process for the immobilization of PDBA-modiﬁ ed

molecules, including nucleic acids and proteins.

Complex formation between PBA or PDBA group and the SHA group occurs in a wide

range of buffer types from pH 5 to 9, and it can tolerate high salt conditions (to 1.5 M) or the

presence of moderate amounts of water-miscible solvents, detergents, chaotropes, and denatur-

ing agents commonly used when working with protein solutions.

Ring formation between SHA groups coupled to solid phase matrices and ligands containing

either PBA or PDBA groups have been investigated for the immobilization of afﬁ nity molecules

for protein puriﬁ cation (Wiley et al., 2001). Using the dimeric PDBA group, immobilization of

modiﬁ ed alkaline phosphatase onto SHA-agarose resulted in good retention of enzyme activity

plus high stability of the coupled protein. The immobilized protein also was stable to elution con-

ditions of acidic (pH 2.5) or basic (pH 11) buffers without cleavage of the boronic acid complexes

or leakage of coupled protein. This indicates that multivalent attachment points using PDBA

instead of PBA result in high tolerance for extreme environmental conditions without hydrolysis.

Bergseid et al. (2000) also reported on the use of SHA-activated chromatography supports

for the coupling of boronate-containing afﬁ nity ligands. In this case, immobilized RNase A was

used to purify anti-RNase antibodies from antiserum samples. RNase A was modiﬁ ed with an

NHS–PDBA crosslinker at a molar ratio of 100:1 (crosslinker:protein), puriﬁ ed to remove excess

crosslinker, and then coupled to an SHA-agarose support in 0.1 M sodium bicarbonate, pH 8.0.

Prolinx originally developed the technology utilizing the SHA–PBA interaction and later por-

tions of it were commercialized by Invitrogen and then Lonza (Cambrex; Versalinx reagents).

A number of PDBA crosslinking agents now are available to introduce the diboronic acid group into

biomolecules. These include heterobifunctional compounds containing an NHS ester (amine reac-

tive), a hydrazide group (aldehyde reactive), a maleimide group (thiol reactive), a pyridyl disulﬁ de

group for reversible linkage to thiols or for thiolation of target molecules, and an iodoacetyl group

for creating thioether bonds. SHA crosslinkers include a hydrazide-containing compound, an NHS

ester, and a bis-SHA compound containing a hydrazide group. Some examples of these reagents are

shown in Figure 17.8 .

Figure 17.7 PBA derivatives react with salicylhydroxamate derivatives to form 5-membered or 6-membered

ring structures, with the 6-membered ring the major product.

3. Boronic Acid–Salicylhydroxamate Reagent Pairs 677

678 17. Chemoselective Ligation: Bioorthogonal Reagents

Conjugation reactions between phenylboronates and salicylhydroxamates entail the forma-

tion of a low afﬁ nity interaction involving a reversible heterocyclic ring formation. Conjugates

formed as a result of this reaction are relatively stable provided the pH is maintained within

the optimal range and there are no competing species in solution, which may exchange for the

SHA group. This may include sugars or carbohydrates containing diols or other organic con-

stituents mentioned previously. The creation of dimeric or multivalent interactions with two or

more SHA groups and a phenylboronate-modiﬁ ed molecule (using PBA or PDBA) will dramat-

ically increase the stability of the linkage over that observed with a single PBA–SHA linkage.

Wiley et al. (2001) found that modiﬁ cation of alkaline phosphatase with PBA or PDBA for

subsequent immobilization on an SHA-agarose support resulted in stable immobilization if

6 PBA groups were present on each alkaline phosphatase molecule or the equivalent of 3 dimeric

PDBA groups. This difference reﬂ ects the dimeric nature of the PDBA group in forming more

than one boronate ring structures per modiﬁ cation.

Figure 17.8 Heterobifunctional crosslinking agents containing the PBA or salicylhydroxamate group for chem-

oselective conjugation purposes.

The use of the PBA–SHA conjugation in a bioorthogonal mode probably will be problem-

atic due to the potential side interactions that could occur involving the phenylboronate groups

with biological molecules in cells or cell lysates. In addition, there are no reports of using SHA

or PBA bio-monomer analogs (e.g., amino acids containing these groups) to incorporate into

biopolymers in vivo as there are with other chemoselective reacting groups. However, the use

of the PBA–SHA reaction as a chemoselective immobilization process for attaching proteins or

other molecules to surfaces may have advantages over other bioconjugation reactive groups,

because the reactive groups are very stable and won ’t hydrolyze in aqueous solution. In addi-

tion, the PBA–SHA reaction can be used with success for the conjugation of two pure biomol-

ecules, such as in the creation of a protein–protein conjugate (Le Roch et al ., 2000).

The following protocol describes the immobilization of a protein on an amine-surface using

the P(D)BA–SHA chemistry.

Protocol

Preparation of P(D)BA-Modiﬁ ed Protein

1. Dissolve a ﬁ rst protein to be modiﬁ ed at a concentration of 1–10 mg/ml in 0.1 M sodium

bicarbonate buffer, pH 8.5. PBS buffer at physiological pH also may be used as the reac-

tion medium.

2. Add a quantity of PDBA–NHS to the protein solution to provide the desired molar

excess of crosslinker over the protein. A suggested starting point is to use a 10- to

15-fold molar excess of reagent, but the optimal amount to be added should be deter-

mined by experimentation to provide a ﬁ nal conjugate having the best possible proper-

ties for the intended application. The PDBA-NHS may be ﬁ rst dissolved in DMF as a

concentrated stock solution and then an aliquot added to the reaction mixture. Mix well

to dissolve.

3. React for at least 1 hour at room temperature with gentle mixing.

4. Purify the modiﬁ ed protein by gel ﬁ ltration or dialysis using a molecular weight cutoff

that will be appropriate for the protein being modiﬁ ed.

Preparation of SHA-Modiﬁ ed Surface

An amine-containing surface, such as an APTS-modiﬁ ed glass slide (see Chapter 13, Section 2),

may be modiﬁ ed with a long-chain NHS-salicylic acid methyl ester derivative (Lonza), which

then can be converted to the SHA group for coupling to a P(D)BA-modiﬁ ed protein. The fol-

lowing protocol describes this method.

1. Thoroughly wash the amine-slide with D.I. water and then with 0.1 M sodium bicarbon-

ate, pH 10 (coupling buffer).

2. Add a quantity of NHS-salicylic acid methyl ester reagent dissolved in coupling buffer to

the slide surface to provide at least a 2-fold molar excess of crosslinker over the quantity

of amines present on the surface. The surface of the slide may be coated with a minimum

solution volume of the crosslinker by layering the solution over the surface. Slide masks

or gaskets may be used to isolate only certain regions for modiﬁ cation. Alternatively,

the slide may be immersed in the crosslinker solution. The NHS-salicylic acid methyl

ester may be ﬁ rst dissolved in DMF as a concentrated stock solution and then an aliquot

3. Boronic Acid–Salicylhydroxamate Reagent Pairs 679