REVIEW Chemical conjugation of biomacromolecules

Chemical Papers 64 (6) 683–695 (2010) DOI: 10.2478/s11696-010-0057-z

REVIEW

Chemical conjugation of biomacromolecules: A mini-review Pavol Farkaš*, Slavomír Bystrický

rc op y

Institute of Chemistry, GLYCOMED Centre, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia Received 4 March 2010; Revised 5 May 2010; Accepted 15 June 2010

ho

Biological studies showed that assembles of biomolecules can dramatically change their physiological effectiveness. Covalent coupling of different types of biomolecules leads to novel biomacromolecules of different properties. Generally, bioconjugate chemistry opens a new dimension in biomedical and biotechnology research. In this review, some important chemical methods of bioconjugates preparation used in the practice are described. Proteins and saccharides modification methods and employment of linkers used to achieve new functionalities are discussed. Common bioconjugation methods are emphasized and novel methods from recent years are described. Except in chemistry, benefits and limits of the studied methods are outlined. c 2010 Institute of Chemistry, Slovak Academy of Sciences Keywords: bioconjugation, chemical ligation, coupling, glycoconjugate

Introduction

A

ut

Bioconjugate preparation is a permanently developing field of organic, bioorganic (Pozsgay & KublerKielb, 2008; Sletten & Bertozzi, 2009), polymer chemistry, or chemistry of materials (Lutz & Börner, 2008; Canalle et al., 2010). So, it is difficult to follow modern trends and applications. Examples of important applications from recent years are remarkably attractive glycoconjugate vaccine studies. Effective long term production of protective saccharide specific antibodies is justified only by a chemical construct containing a saccharide antigen and a protein carrier (Pozsgay & Kubler-Kielb, 2008). The goal of bioconjugate chemistry is to develop simple and efficient methods for chemical bonding of biomolecules with other molecules. For this purpose, a pre-required preceding modification of the biomolecule(s) using bifunctional reagents is often necessary. These reagents are used as linkers, and they might bring new functionalities into molecules, or they act just as spacers improving spatial availability of functional groups. Functional modification of biological molecules with a high degree of selectivity or specificity is needed. A good method of functional modification should be generally applica*Corresponding author, e-mail: [email protected]

ble to a wide range of biomolecules. Up to day, no such universal coupling technique has been developed.

Methods for selective functionalization of biomacromolecules Proteins can be chemically modified using a large range of methods (Hermanson, 1996; Sletten & Bertozzi, 2009; Canalle et al., 2010). This chapter is focused on those methods successfully used to modify proteins and saccharides. Generally, chemical reactivity of proteins depends on side chains of their amino acids, on their N- and Cterminal residues (Fig. 1) and on their spatial structure. Crucial side chains from the reactivity view point are γ-carboxyl group of glutamic acid, β-carboxyl group of aspartic acid, thiol group of cysteine, and ε-amino group of lysine. Among them, the thiol of cysteine is the most potent nucleophilic group, followed by the amino and hydroxyl ones. Hydroxyl groups have similar pKa as water and they are generally unreactive in aqueous solutions. The imidazolyl group of histidine, thioether moiety of methionine, indolyl group of tryptofan, phenol group of tyrosine, guanidyl group of arginine, and aliphatic hydroxyl group of ser-

684

P. Farkaš, S. Bystrický/Chemical Papers 64 (6) 683–695 (2010)

7.6–8.0 7.6-8

6.7–7.1 6.7-7.1

NH3

H N

8.8–9.1 8.8-9.1 SH

N OOC

COO 3.7–4.5 3.7-4.5

2.1–2.4 2.1-2.4

HN H2N

NH2 >>12 12

NH3 9.3–10.5 9.3-10.5

OH 9.7–10.1 9.7-10.1

rc op y

Fig. 1. Typical functional groups of proteins and their pKa values (Hermanson, 1996).

1,5-difluoro-2,4-dinitrobenzene (DFDNB), 3,4-diethoxycyclobut-3-ene-1,2-dione (diethyl squarate, DES), and thiophosgene (TP). Based on specific criteria, i.e., cost, reactivity, sugar loading, and homogeneity, DSS, DFDNB, and DES seem to have advantages over the other cross-linking reagents (Izumi et al., 2003). Carboxyl group is the most abundant functional group in proteins. Carboxyl moieties can be activated (toward nucleophil, e.g. amine) as acyl esters, acyl azides, acylimidazoles, anhydrides, etc. There are different ways of coupling reactive carboxyl derivatives with an amine to form amide, two of which are commonly used. In the first method, a reactive acylating agent is formed from the acid in a separate step followed by immediate treatment with the amine. The second method consists in the acylating agent being generated in situ from the acid in the presence of the amine by an addition of an activating or coupling agent. A review of such methods and their comparison is available (Montalbetti & Falque, 2005). Activation of carboxyl groups on proteins may lead to intramolecular and intermolecular cross-linking, especially in the first method. Succinylation of amino groups leads not just to an increased number of carboxylic groups, but it can also improve immunological properties of proteins (or their final conjugates) (Pavliakova et al., 1999).

A

ut

ho

ine are of less or no importance (Hermanson, 1996; Sletten & Bertozzi, 2009). Thiol group is a mild nucleophil which can be selectively modified under appropriate reaction conditions. Proteins excessively differ in the number of thiols, e.g. BSA contains only one Cys residue, but KLH contains over 700 thiols from Cys residues (Hermanson, 1996). One of the possibilities of generating thiol groups is reduction of internal disulfide bonds, e.g. with 1,4disulfanylbutane-2,3-diol or a newer reagent N,N dimethyl-N,N -bis(mercaptoacetyl)hydrazine wich reduces disulfide bonds seven times faster at pH 7 (Singh & Whitesides, 1991). To introduce more thiol groups into proteins, they are often derivatized with linkers that contain a thiol group, e.g. 2-iminothiolane, which reacts with an amino group to give a new thiol (Kubler-Kielb et al., 2006) (see Tab. 2). Most common sulfhydryl reactive derivatives are α-halocarbonyl compounds, maleimides, acryloyl derivatives, aziridines, oxiranes, fluorobenzene derivatives, disulfide reagents, etc. (Hermanson, 1996). Important methods are discussed in chapter Thioether bond formation. The ε-amine group of lysine differs in pKa from the primary α-amino terminus by two orders (pKa (ε) 10.2 and pKa (α) 7.8) and is generally the second most abundant functional group in proteins. Most important reactions of amino groups including acylation (formation of amides with active esters), reductive amination, and reaction with squaric acid esters are discussed in next chapters. Other reactive derivatives used for amino group derivatization are e.g. isothiocyanates, isocyanates, acyl halides, azides, imidoesters, anhydrides, carbonate compounds (Hermanson, 1996). Direct cross-linking of mannosyl ethanolamine and the bovine serum albumin protein (BSA) was investigated using various homobifunctional reagents: N,N disuccinimidyl carbonate (DSC), di-(N-succinimidyl) glutarate (DSG), di-(N-succinimidyl) suberate (DSS), ethylene glycolbis(N-succinimidylsuccinate) (EGS),

Derivatization of saccharides Carbohydrates are an extremely wide group of biomolecules consisting of numerous possible monomers and their linkages. Structural monomers can also be acetylated, phosphorylated or branched. Except of hydroxyl groups, carbohydrates often bear carboxyl groups, amino groups and many other reactive groups. For conjugation of smaller saccharides, free aldehyde or ketone of the hemiacetal or hemiaminal group, respectively, is often used in reductive amination. If a saccharide is prepared synthetically, aglycon often bears the required reactive functional group. Polysaccharides, almost in all cases, have to be chemically derivatized before conjugation. Most employed derivatization method is selective oxidation of vicinal diols by periodate (Lees et al., 2006); sec-hydroxyls can be oxidized by 1,1,1-triacetoxy-1,1dihydro-1,2-benziodoxol-3(1H)-one, Dess-Martin periodinane (DMP), to give aldehydes, or by nitrogen oxides or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) to give carboxyl groups (Ďurana et al., 2006). Another types of derivatization are the “activation” of hydroxyls (sterically less hindered hydroxyls) by cyanogen bromide (Amir-Kroll et al., 2003) or 1cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP), both give reactive cyanato derivatives (Fig. 2) (Kohn & Wilchek, 1983), and the multicomponent Ugi reaction (Fig. 3) (García et al., 2009). Acidic hydrolysis of polysaccharides is used to get more sol-

685


Protein

Saccharide

BrCN/(ET)3N or CDAP Saccharide OH

NH2

Saccharide

N O

II

IIII H2O Saccharide

O

H N

O

H2O Protein

NH

OH NH

III III Saccharide

O

IV IV

H N

O

Saccharide

Protein

O

NH2 O

Fig. 2. Saccharidereacts with a cyanylating agent to give cyanato derivative I. Derivate I reacts with the amino group of protein to give a conjugate with imidocarbonate II linkage which is further hydrolyzed to N-substituted carbamate III. I can also undergo hydrolysis to give inert carbamate IV.

CN + O

N

NH2 + Saccharide COOH Protein

Saccharide

NH

O O

Fig. 3. Cross-linking of carboxylic (or e.g. O-carbomethylated) saccharide with acetone, tert-butyl isocyanide, and protein using the Ugi reaction (pH 5, 4 ◦C).

uble and lower molecular mass saccharides (Anderson et al., 1985).

ho

“Classical” coupling methods Reductive amination

ut

Reductive amination is one of the most popular methods for the preparation of glycoconjugates especially from unprotected free mono- and oligosaccharides. Free aldehydes and ketones react with amines (similarly with e.g. hydrazine, hydrazide, oxime derivatives) to form unstable (reversible reaction) imines (Schiff bases). After the reduction of imines, stable sec-amines are formed; this two step reaction sequence is called reductive amination. The initially formed imine (still in equilibrium with the starting aldehyde/ketone and amine) is in the second step converted (usually in a one pot reaction) to a stable secondary amine using a reducing agent. As reducing agents, hydroborates, of which Na(BH3 )CN is most common, are generally used due to their high selectivity to imines and relative unreactivity to oxo-groups. Recently, a non toxic alternative of Na(BH3 )CN was introduced and studied (from simple mono- to nonasaccharide) with a new agent, NaBH(OAc)3 , reactivity of which is comparable with Na(BH3 )CN (Dalpathado et al., 2005). In the case of saccharides, the Amadori rearrangement can complicate this reaction. On the other side, the Amadori rearrangement is proposed to be responsive in interesting high temperature glycosylation of proteins with carbohydrates (Boraty´ nsky & Roy, 1998). If a carbohydrate molecule does not contain an

A

aldehyde (or hemiacetal) group, a linker/spacer with a protected aldehyde/ketone group, e.g. acetals (Farkaš & Bystrický 2007, 2008; Zhang et al., 1998), enes (ozonolysis) (Bernstein & Hall, 1980; Xue et al., 2002), has to be used to achieve aldehyde as a functional partner. Vicinal hydroxyl group oxidation resulting in aldehydes is often used in the case of polysaccharides (Lees et al., 2006). Unfortunately, unselective oxidation gives random distribution of aldehydes and may damage molecule epitopes. Simple hydrolysis of polysaccharide to oligosaccharides that can be directly attached to proteins via reductive amination is a better choice (Anderson et al., 1985). An interesting example of this method is direct conjugation of meningococcal lipooligosaccharides to the tetanus toxoid (TT) protein (Mieszala et al., 2003). Two different oligosaccharides V and VI obtained by chemical degradation of the original L7 lipooligosaccharide were compared (Fig. 4).

rc op y

Protein

Amide bond formation Amide bond formation is the reaction mostly used to introduce various linkers or spacers to biomolecules to be coupled, especially in the case of proteins. For a review on amide bond formation methods see Albericio (2004), Montalbetti & Falque (2005). Active esters Carbodiimide based activation of carboxyl groups is the most common method for active esters preparation. N-substituted carbodiimides are widely used in peptide synthesis; in bioconjugate chemistry, water soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) has found general usage (Grabarek & Gergely, 1990; Bauminger & Wilcheck, 1980; Hermanson, 1996). Activation of carboxylic acids with carbodiimides is complicated by hydrolysis and irreversible intramolecular rearrangement of the desired reactive O-acylisourea derivative to the unreactive N-acylurea derivate (Bauminger & Wilcheck, 1980). The latter complication is often overcome using one pot formation of active ester by an addition of an auxiliary nucleophil, e.g. 3-sulfo1-hydroxysuccinimide (S-NHS) or 1-hydroxy-1,2,3-

686


Saccharide O HO

OH

OH

COOH

NH2

Protein

O HO

Saccharide

Saccharide O HO

OH

OH

OH OH COOH

HO

COOH

HO

O HO

N

O

V V

Protein

(lipid A)-Saccharide Saccharide

O

O HO

O

HO HO

OH

COOH

HO

NH

HN

VI VI

rc op y

acyl

OH OH

Protein

Fig. 4. Oligosaccharides V and VI obtained by acid hydrolysis and O-deacylation of L7 lipooligosaccharide, respectively.

O Peptide

O

S

O

R

Peptide

HS + SH

COOH

Peptide

+ H2N

Peptide′

S

HS

H2N

NH2

HS

Peptide′

Peptide

ho

Peptide e′

+

O

COOH

S

N H

Peptide′

Fig. 5. Native chemical ligation of two peptide fragments using a thiol-catalyst (R = alkyl).

A

ut

benzotriazole (HOBt), simultaneously with carbodiimide (Grabarek & Gergely, 1990; Bulpitt & Aeschlimann, 1999). Nowadays, newer reagents forming active esters directly in one step, e.g. 4-(4,6-dimethoxy1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Schlottmann, et al., 2006; Farkaš & Bystrický, 2007), are available. Native chemical ligation

Native chemical ligation (NCL) of unprotected peptide segments was developed by Dawson et al. (1994) and demonstrated on one step preparation of cytokine containing multiple disulfides. NCL is a reaction between a peptide containing C-terminal thioester and another peptide containing N-terminal cysteine. The reversible first step is followed by a S→N acyl transfer yielding an amide (‘peptide’) bond at the ligation site. No protecting groups are required and the reaction occurs in water at physiological pH. α-Alkylthioesters are usually used because of their simple preparation, they are, however, almost unreactive or they react very slowly. For this reason, additional catalysts are used for the in situ transthioesterification. 4-Mercaptophenylbenzoic acid (Fig. 5) has shown good accuracy in performing ligations in short reaction time (hours) and high yields (Johnson & Kent, 2006).

Decarboxylation condensation Decarboxylation condensation originally developed for peptide synthesis is a possible method for the conjugation of two biomolecules containing appropriate functional groups. The reaction between ketocarboxylic acid and N-hydroxyamino acid results in an amide bond via a nitrilium intermediate (Fig. 6). This reaction proceeds in polar protic and aprotic solvents, requires no reagents or catalysts, produces only water and carbon dioxide as byproducts, and readily tolerates unprotected functional groups. This method has the potential of a novel chemoselective ligation method for the synthesis of peptides and other complex materials – bioconjugates (Bode et al., 2006). A more detailed reaction mechanism was also investigated (Sanki et al., 2009). Thioether bond formation α-Halocarbonyl and thiol derivatives react forming conjugates with a thioether bond. It is an irreversible SN 2 reaction which is not selective and other possible nucleophiles may also react. Observed byproducts are S-carbonyl derivatives and dialkylation products. Thiol derivatives react fastest at pH 9–10 (e.g. amines react at pH 7.5–9; histidine at pH 7; thiol at pH 1.7) (Gurd, 1967).

687


O

HO

OH +

Peptide

+ H+

N Peptide′ H

O

R

−CO2 −2 H2O

O

H2O

N R′

−H

+

Peptide

Peptide′

N H

Fig. 6. Reaction of the N-hydroxyamino derivative of peptide with the α-ketoacid derivative of peptide (glycopeptide).

H N

N

SAc +

n

H N

Saccharide

H N

O

H N

Saccharide

O

O 4

n

O

O

Saccharide

O

Protein

H N

H N

S

O

H N

+ HS

H N

N

S

O

b Protein

NH

O

O

Br + AcS

n

Protein

O

O

Protein

H N

op y

Protein

O

O

a

O

Saccharide

O

Saccharide

O

O

O

n

rc

Protein

H N

O

S

4

N H

O

Saccharide

O

ut ho

Fig. 7. Thioether bond formation between functionalized saccharide and protein. S-acetylthiol linker is deprotected by excess addition of hydroxylamine during the coupling reaction.

A

N-Maleimides are selective Michael acceptors of amine nucleophiles, especially below pH 7; however, at pH > 8, they prefer amine addition. Both maleimide and halocarbonyl reactions are complicated by the competition of thiol groups and maleimide with hydrolysis (which is negligible at pH 7). A comparison of model reactions of thiol with N-ethylmaleimide (NMM) at pH 6, and 2-iodoacetic acid (IAA) or 2iodoacetamide (IAM) at pH 8 showed that NMM reacts significantly faster and the rate difference is even greater when IAA or IAM reacts at pH 6 (Rogers et al., 2006). Similar results were observed by Grandjean et al. (2009), where the authors conjugated an acid hydrolyzed O-specific polysaccharide (O-SP) of Vibrio cholerae to TT or BSA proteins (Fig. 7a). Linking O-SP through maleimide (pH 8) in comparison to bromoacetyl linking (pH 6) was more effective (Table 2). Conjugation of ganglioside antigens with thiol aglycon to BSA modified with an acrylate linker based on water soluble triethylene glycol was tested (Fig. 7b) (Dziadek et al., 2008). In a similar case, peptide bearing N -terminal mercaptopropionic acid residue was conjugated to the antigenic manotriose bearing water soluble triethylene glycol acrylate (Xin, et al., 2008). Photochemically or thermally induced radical addition of thiol to alkene (Griesbaum, 1970; Hoyle & Bowman, 2010) can be considered to be bioorthogonal (Jonkheijm et al., 2008). Its usage was demonstrated by conjugation of glycopeptide MUC1 antigens and a protein carrier (Wittrock et al., 2007).

Activation of hydroxyl groups Formation of reactive cyanate ester derivatives of polysaccharides was firstly achieved through a reaction using hazardous cyanogen bromide (BrCN). Generally, cyanate esters may undergo hydrolysis to form inert carbamate. In situ formation of highly unstable, but reactive N-cyano-triethylammonium bromide (BrCN/(Et)3 N) leads to a very effective activator of polysaccharides; however, the reactive complex cannot be isolated since it decomposes at above 0 ◦C. A cyanylating agent based on a stable tetrafluoroborate complex, CDAP, with improved activation yields, 7– 20 times higher than for BrCN (Kohn & Wilchek, 1983), was introduced into the chemistry of polysaccharides. CDAP activated dextran was reacted with amine and hydrazine reagents at pH 4–9. Derivatization with amines was pH dependent with a minimum at pH 5 and a maximum at pH 9.3. In contrast, derivatization with hydrazides was almost pH independent in the pH range of 4–9 (Shafer et al., 2000). Similarly, the conjugation of yeast cell surface mannans with protein human serum albumin (HSA) was examined. Direct conjugation yielded soluble products, no cross-linking was observed under the chosen reaction conditions (pH 8, 4 ◦C, quenching by ethanolamine) (Bystrický et al., 2000).

688


Recently developed coupling or ligation methods Alkyne-azide coupling

Staudinger ligation Staudinger ligation (SL) as a reaction of azides and phosphines is a modification of the Staudinger reaction (Staudinger & Meyer, 1919) with an electrophilic trap on triarylphosphine. It was introduced and firstly used for bioconjugation by Saxon and Bertozzi (2000). Aza-ylide contains a highly nucleophilic nitrogen atom which may be intercepted by almost any kind of electrophil. Aza-ylide intermediate rearranges in aqueous media forming an amide and a phosphine(V) oxide in the so called Staudinger reduction. If an electrophilic group is placed near a reactive nitrogen atom, a covalent amide bond can be generated prior to hydrolysis. A study on the Staudinger ligation mechanism established a platform for its expanded use in biological applications (Lin et al., 2005, Kiick et al., 2002). Staudinger ligation has even found application in the complex environment of living cells (K¨ ohn & Breinbauer, 2004) as the two reaction partners are bioorthogonal to almost all functionalities in biological systems and they react at room temperature in appropriate aqueous environment. Despite these findings, so called ”traceless” SL is a method of choice nowadays. The main advantage is that no phosphine(V) linker is build in the conjugate. As shown in Fig. 9d, only amide bond is formed between the conjugated molecules. Less efficacious coupling reagents and reaction conditions lead to the accumulation of an amine byproduct (resulting from the Staudinger reduction) or a phosphonamide byproduct (resulting from the aza-Wittig reaction) as shown in Table 1 (Soellner et al., 2006). Acid hydrolyzed O-SP of Vibrio cholerae derivatized with azide linkers were conjugated to TT and BSA proteins with phosphine(5) linkers. No significant differences were observed between the conjugation with TT or BSA, not even when using azide link-

A

ut

ho

rc op y

Azides and alkynes, due to their weak acid-base properties, are almost inert towards biomolecules; so far it is simple to introduce them into molecules using a linker. Cu(I)-catalyzed azide-alkyne [3 + 2] cycloaddition can be conducted in many solvents and mixtures of water and (partially) miscible organic solvents. Nevertheless, water is still the most popular reaction solvent providing highest rates and yields. As a source of Cu(I), its in situ preparation from common Cu(II) salts, e.g. CuSO4 , with ascorbic acid/Na-ascorbate as a reductant is preferred (Rostovtsev et al., 2002). In some cases, where it is impossible to use ascorbic acid, Cu(II)/Cu redox system is a suitable Cu(I) source. Addition of tris-(benzyltriazolylmethyl)amine (TBTA) (Fig. 8) greatly enhances the reaction rate (Wang et al., 2003). TBTA was shown to be a powerful stabilizing ligand for Cu(I) protecting it from oxidation and disproportionation while enhancing its catalytic activity (Chan et al., 2004). TBTA derivatives were found to be the best accelerating ligands for Cu(I)-catalyzed azide-alkyne cycloaddition reactions. A water-soluble ligand, potassium 5,5 ,5 -(2,2 ,2 nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)) tripen tanoate (BimC4 A)3 (Fig. 8), was found convenient for rapid and high-yielding synthesis of several functionalized triazoles (Rodionov et al., 2007). A Cu-free variant of the reaction was developed to avoid the cytotoxicity of copper. The reagent is not a terminal alkyne but the smallest cyclic alkyne, cyclooctyne, in which the ring strain promotes the reaction. More potent reagents contain an electron-withdrawing fluorine substituent, 3,3difluorocyclooctyne (DIFO, Fig. 8). Derivatives of DIFO and the terminal alkyne were conjugated to azido-homoalanine labeled recombinant dihydrofolate reductase (DHFR), showing their similar reactivity (Baskin et al., 2007). Another comparison, labeling of living cells bearing azidosialic acid, showed that the DIFO reagent is superior to the terminal alkyne and phosphine (Staudinger ligation reaction partner)

(Baskin et al., 2007). Problem with hydrophobicity may be overcome by 6,7-dimethoxyazacyclooct-4-yne (DIMAC) whose water solubility and polarity is superior to those of DIFO derivatives (Sletten & Bertozzi, 2008). More information and applications of azidealkyne click chemistry were recently reviewed (Lutz & Zarafshani, 2008; Sletten & Bertozzi, 2010).

(CH2)4COOK N

N N Ph TBTA

MeO F

N

N

N

F

MeO N

3

3 (BimC4A)3

N

HOOC O DIFO

COOH

O DIMAC

Fig. 8. Structure of additives used in Cu(I)-catalyzed azide-alkyne [3 + 2] cycloaddition and structure of DIFO derivative with the suggested substituent (grey) used by Baskin et al. (2007).

689


a

P

P

N N N

P

N

N

P

- N2

N N

N N

N

P

H2N +

b

N

H2O

P O

c

O

O N3

O R

N H P Ph Ph O

H2O

Ph

P

R′

d

R

Ph

R′

O R

S

O

O

N3 R′ BH3 Ph H2O P R

N H

Ph

R′ + HS

P

Ph

Ph

a

O

H N

Protein

22

rc op y

Fig. 9. (a) Formation of ylide from azide and phosphine. In the presence of water (b) the reaction continues to form amine and phosphine(V) oxide in the so called Staudinger reaction, or Staudinger reduction. If there is a suitable electrophil, the reaction continues toward the Staudinger ligation. Example of a “nontraceless” Staudinger ligation (c) and a “traceless” Staudinger ligation (d) employing VIII-like and X, respectively.

O

N

N H

+

O O

O Protein N H

O

(NH)2

N (CH2)3

O

ho

Saccharide

Saccharide

O

O Saccharide (CH2)5

O

O

H N

N H

2

XII XII

Saccharide

O

XIII XIII

ut

O

O

2

O

O

b

H N

A

Fig. 10. (a) Preparation of hexasaccharide and human serum albumin (HSA) protein glycoconjugate by the Diels-Alder cycloaddition. (b) Comparison of the aglycon-linkers XII and XIII coupling rates.

Table 1. Effect of a coupling reagent on the relative rate and yield of amide between the amine and phosphonamide byproducts (DMF/H2 O, 6 : 1) (Soellner et al., 2006) Reagent

HO

PPh2

HO

krel

Amide/%

VII

1

11

VIII

8.5

38

IX

5.5

39

X

64

95

XI

62

99

PPh2 HS HS

PPh2 PPh2

HS PPh2

ers with different lengths (Grandjean et al., 2005). Diels–Alder 2+3 cycloaddition Diels–Alder-type cycloadditions, a part of pericyclic reactions, are a very important class in organic chemistry. In 1980, Rideout and Breslow (1980) uncovered an interesting and very important fact that the “hydrophobic effect” can dramatically increase the rate of this reaction. This phenomenon is exploited in many organic reactions (Li, 2005). First reported use of DA cycloaddition in the preparation of bioconjugates describes conjugation of a monosaccharide equipped with diene algycon and maleimide linker derivatized HSA. pH optimization showed that an addition reaction in phosphate-borate favors lower pH, best results were obtained at pH 5.7, but reaction efficiency in ion-exchanged water was at the same level. A comparison of two linkers XII and XIII of different lengths showed a correlation of the distance between the more hydrophilic saccharide moi-

690


O HO

O

OH

HO

OH Saccharide O HO

EtO NH2

H N

OH

OEt pH 7

OH

Saccharide O HO

O

H N

O

O

N H

OEt

O

Protein, pH 9 HO

OH OH

Saccharide O HO

H N

O

O

N H

N Protein H

O

O

O Protein

O

S

N H

N H

+ O

NH2

Protein

H N

H N

O

S

N H

H N

Saccharide

O

O

O

O

O Saccharide

rc op y

Fig. 11. Use of DES for Streptococcus oligosaccharide antigen and protein coupling via diethyl squarate.

O

N H

N H N

O

O

Fig. 12. Ketohexanoyl derivative of the hexasaccharide reaction with aminooxylated BSA.

ut

Squaric acid chemistry

ho

ety and the diene, and of the reaction rate (Fig. 10b). Utility of this method was proved by linking a hexasaccharide (half repeating unit of Shigella dysenteriae type 1 polysaccharide) to HSA (Fig. 10a). After eight hours, incorporation reached an average of 13 molecules per HSA (Pozsgay et al., 2002).

A

Squaric acid chemistry was first used by Tietze et al. (1991) to conjugate p-aminophenyl glycosides to protein (BSA) using diethyl squarate (DES). In the first step, nucleophil (amine group of hapten or a molecule to be conjugated) reacts with electrophilic squarate by the addition-elimination mechanism at pH 7 to give a monosubstituted product. At pH 9, substitution of the second electrophilic group takes place. Conclusions based on the experiments with a large group of glycosides conjugation with BSA were that the conjugation of hydrazide haptens is generally slower and less efficient than that of amine. No advantage of the use of any of the four tested dialkyl squarate esters (methyl, ethyl, butyl, and decyl) was found. Methyl ester undergoes the fastest hydrolysis in an aqueous solution, pH 9 (Hou et al., 2008). Capsular polysaccharide of type III group B Streptococcus depolymerized by ozonolysis to the molecular size of 20 kDa was peracetylated and then transformed to its aminoderivative by 1,2-ethylenediamine on the reducing end. Such oligosaccharide antigen was coupled to TT to give a conjugate with two oligosaccharide chains on TT in average (Fig. 11) (Wang et al., 2002).

Aminooxy-aldehyde/ketone condensation This approach is based on the well known oxime formation between O-alkyl hydroxylamines and aldehydes/ketones. It may be, among many other advantages, a suitable substitution of reductive amination where basic conditions can cause hydrolysis e.g. de-Oacylation. Various L-rhamnose derivatives, with aldehyde or ketone groups on the aglycon were subjected to conjugation with aminooxylated BSA (Fig. 12). The number of bound haptens was very similar for all linkers in the pH range from 5.5 to 7.0. A little lower number of bound haptens was found in the case of D-ribitolphosphate hapten (Kubler-Kielb & Pozsgay, 2005). In another case, preparation of Bacillus anthracis polyγ-D-glutamic acid–protein conjugates by oxime chemistry, hydrazone bond formation and thioether were compared, see Table 2. (Kubler-Kielb et al., 2006). More applications of oxime chemistry in bioconjugation were described by Lees et al. (2006); e.g., functionalization of proteins and polysaccharides with aminooxy groups and aldehyde groups.

Conclusions There are various methods which are less or more generally used for bioconjugate preparation. A combination of these methods and reasonable utilization of linkers with the desired functional properties may be applied to the conjugation of almost all biomolecules. Many applications such as decarboxylation condensation or native ligation were developed and applied just

Azide–alkyne coupling

Thioether bond formation, radical thiol–ene coupling

Thioether bond formation, addition reaction

Thioether bond formation, nucleophilic substitution

Reductive amination

Coupling method

ut

A

Maltotriose Maltotriose Lipooligosaccharide, V Lipooligosaccharide, VI Dextran, 9.3 kDa Maltotriose Polysaccharide, 6 kDa Polyglutamic acid—Br Polyglutamic acid—SH Polysaccharide—SAc Polysaccharide—SAc Tetrasaccharide—SH

TT—NH2 BSA—NH2 TT—NH2 TT—NH2

TT—(L)—Br TT—(L)—Br TT—(L)—Br TT—(L)—SH TT—(L)—Br TT—(L)—Br BSA—(L)—Br BSA—(L)—Br

glycopeptide—— — glycopeptide—SH

Dye—yne Dye—N3

BSA—(L)—SAc BSA—(L)—— —

CPMV—N3 CPMV—yne

Coupling conditions

11.2 (43) 21.4 (82) 16.0 (73) 11 16 10 (30) 2 (7) 8 (53)

6.0–20 9.9 18.9 15.5

60 (100) 48 (80)

9 (38) 8 (32)

8.5 8.5 8.5 7.4 7.4 8.3 8.3 7.8

4 ◦C; pH 8 4 ◦C; pH 8

20 ◦C; ACVA; hν hν

16 h 16 h

8h

8h 2h 2h

2d 2d o.n. 1.5 h 1.5 h 30 h 30 h o.n.

7–14 d 14 d 4d 4d

Time

94–96 80

n.a. n.a.

∼ 100 48b 72

n.a. n.a. ?a n.a. n.a. 57 53 n.a.

n.a. n.a. 36? 36?

Yield /%

(+) Selectivity, % of derivatization, yield (–) additives.

(+) reaction time, selectivity (–) co-initiator, or high temperature

(+) Simple purification, high yield, reaction time

(+) Efficiency

(+) Simple chemistry (–) Not universal, long reaction time

Merits/(+) Handicaps/(–)

rc op y

pH pH pH pH pH pH pH pH

pH 8.9 r.t.; pH 6 r.t.; pH 6

r.t.; r.t.; r.t.; r.t.; r.t.; r.t.; r.t.; r.t.;

r.t.; pH 8.0 r.t.; pH 8.0 37 ◦C; pH 9 37 ◦C; pH 9

ho

Molar ratio (Reacted groups/%)

BSA—(L)—— 8 — Oligosaccharide—SH, Fig. 7b TT—(L)—SAc Oligosaccharide—maleimide, Fig. 7a 14 BSA—(L)—SAc Oligosaccharide—maleimide 4

Hapten and linker f. group

Protein

Wang et al. (2003) Wang et al. (2003)

Wittrock et al. (2007)

Dziadek et al. (2008) Grandjean et al. (2009) Grandjean et al. (2009)

Pawlowski et al. (1999) Pawlowski et al. (1999) Pawlowski et al. (2000) Kubler-Kielb et al. (2006) Kubler-Kielb et al. (2006) Grandjean et al. (2009) Grandjean et al. ( 2009) Leung et al. (2009)

Pawlowski et al. (1999) Pawlowski et al. (1999) Mieszala et al. (2003) Mieszala et al. (2003)

Reference

Table 2. Selected coupling methods, molecule per protein substitution comparison; substitution is expressed as a mole ratio, and as the percentage of coupled linker functional groups


691

Oligosaccharide—N3

Monosaccharide, Fig. 12 Tetrasaccharide Polyglutamic acid—CHO

BSA—(L)—COR BSA—(L)—COR rPA—(L)—ONH2

2

(+) Short reaction time, high yield. Stability of borane-protected phosphane linker.

r.t.; pH 9

22 ◦C

2d

8h 24 h

n.a.

n.a. n.a.

Grandjean et al. (2005)

Reference

(+) Simple chemistry, recovery of unreacted material

Wang et al. (2002)

(+) High derivatization, Pozsgay et al. (2002) uncoupled saccharides can Berkin et al. (2002) be recovered

rc op y

70

4h

40–45 ◦C

Merits/(+) Handicaps/(–)

37

r.t.; pH 7.4

o.n.

n.a.

(+) Hydrazides reacts faster then amines

Kubler-Kielb et al. (2006)

17–21 (50–62) 37 ◦C; pH 5.5–7 12 h ∼ 85b (+) Stable oxime bond, in Kubler-Kielb and Pozsgay, 2005) contrast to reductive Kubler-Kielb and Pozsgay, 2005) 17 (50) 37 ◦C; pH 6.8 12 h ∼ 85b 22 r.t.; pH 7.4 o.n. n.a. amination, which forms Kubler-Kielb et al. (2006) unstable Shiff base

rPA —(L)—CHO Polyglutamic acid—CONHNH2

Oligo—OEt, Fig. 11

TT—NH2

13 (59) 26 (68)

7.3 (59)

Time Yield /%

Coupling conditions

ho

Molar ratio (Reacted groups/%)

ut

Hapten and linker f. group

BSA—maleimide Hexasaccharide—diene, Fig. 10a HSA—maleimide ManNAc—diene

TT—P(V) (Fig. 9g)

Protein

a) Recovery of the protein was typically 95 % and efficiency of the conjugation estimated by a comparison of the protein bromoacetyl groups and the actual conjugate substitutions was about 80 %; b) yield calculated to a protein; n.a. = not available; o.n. = over night; CPMV – virus; rPA, recombinant B. anthracis protective antigen.

Hydrazone bond formation

Oxime bond formation

Squarate chemistry

Diels–Alder cycloaddition

Staudinger L.

Coupling method

Table 2. continued

A

692 P. Farkaš, S. Bystrický/Chemical Papers 64 (6) 683–695 (2010)


in protein synthesis up to date. Their possibility to be used widely is unarguable. Acknowledgements. This work was supported by APVV 0032-06 and VEGA 2/0040/10 Grants of the Slovak Grant Agencies.

Abbreviations Acetyl 4-[2-(3-Carboxy-1-cyano-1-methylpropyl)diazen-1-yl]-4-cyano-4-methylbutanoic acid (4,4 -Azobis(4-cyano valeric acid)) (BimC4 A)3 Potassium 5,5 ,5 -(2,2 ,2 -nitrilotris(methylene)tris(1H-benzimidazole-2,1diyl))tripentanoate BSA Bovine serum albumin CDAP 1-Cyano-4-(dimethylamino)pyridinium tetrafluoroborate CMPV Cowpea mosaic virus DA Diels–Alder DES Diethyl squarate DFDNB 1,5-Difluoro-2,4-dinitrobenzene DIFO 3,3-Difluorocyclooctyne DIMAC 6,7-Dimethoxyazacyclooct-4-yne DMF Dimethyl formamide DMTMM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium chloride DSS Di-(N-succinimidyl) suberate EDCI 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide HSA Human serum albumin IAA 2-Iodoacetic acid IAM 2-Iodoacetamide L Linker NCL Native chemical ligation NMM N-Ethylmaleimide O-SP O-Specific polysaccharide SL Staudinger ligation TBTA Tris-(benzyltriazolylmethyl)amine TT Tetanus toxoid

104, 16793–16797. DOI: 10.1073/pnas.0707090104. Bauminger, S., & Wilcheck, M. (1980). The use of carbodiimides in the preparation of immunizing conjugates. Methods in Enzymology, 70, 151–159. Berkin, A., Coxon, B., & Pozsgay, V. (2002). Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A. Chemistry – A European Journal, 8, 4424–4433. DOI: 10.1002/1521-3765(20021004)8:19 3.0.CO;2-1. Bernstein, M. A., & Hall, L. D. (1980). A general synthesis of model glycoproteins: coupling of alkenyl glycosides to proteins, using reductive ozonolysis followed by reductive amination with sodium cyanoborohydride. Carbohydrate Research, 78, C1–C3. DOI: 10.1016/S0008-6215(00)83676-9. Bode, J. W., Fox, R. M., & Baucom, K. D. (2006). Chemoselective amide ligations by decarboxylative condensations of Nalkylhydroxylamines and α-ketoacids. Angewandte Chemie International Edition, 45, 1248–1252. DOI: 10.1002/anie. 200503991. Boraty´ nsky, J., & Roy, R. (1998). High temperature conjugation of proteins with carbohydrates. Glycoconjugate Journal, 15, 131–138. DOI: 10.1023/A:1007067513242. Bulpitt, P., & Aeschlimann, D. (1999). New strategy for chemical modification of hyaluronic acid: Preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels. Journal of Biomedical Materials Research Part A, 47, 152–169. DOI: 10.1002/(SICI)10974636(199911)47:23.0.CO;2-I. Bystrický, S., Machová, E., Bartek, P., Kolarova, N., & Kogan, G. (2000). Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP) – an effective route of antifungal vaccine preparation. Glycoconjugate Journal, 17, 677–680. DOI: 10.1023/A:1011002118819. Canalle, L. A., L¨ owik, D. W. P. M., & Hest, J. C. M. (2010). Polypeptide–polymer bioconjugates. Chemical Society Reviews, 39, 329–353, DOI: 10.1039/b807871h. Chan, T. R., Hilgraf, R., Sharpless, K. B., & Fokin, V. V. (2004). Polytriazoles as copper(I)-stabilizing ligands in catalysis. Organic Letters, 6, 2853–2855. DOI: 10.1021/ol0493094. Dalpathado, D. S., Jiang, H., Kater, M. A., & Desaire, H. (2005). Reductive amination of carbohydrates using NaBH(OAc)3 . Analytical and Bioanalytical Chemistry, 381, 1130–1137. DOI: 10.1007/s00216-004-3028-9. Dawson, P. E., Muir, T. W., Clark-Lewis, I., & Kent, S. B. (1994). Synthesis of proteins by native chemical ligation. Science, 266, 776–779. DOI: 10.1126/science.7973629. Ďurana, R., Lacík, I., Paulovičová, E., & Bystický, S. (2006). Functionalization of mannans from pathogenic yeasts by different means of oxidations—preparation of precursors for conjugation reactions with respect to preservation of immunological properties. Carbohydrate Polymers, 63, 72–81. DOI: 10.1016/j.carbpol.2005.08.003. Dziadek, S., Jacques, S., & Bundle, D. R. (2008). A novel linker methodology for the synthesis of tailored conjugate vaccines composed of complex carbohydrate antigens and specific TH cell peptide epitopes. Chemistry – A European Journal, 14, 5908–5917. DOI: 10.1002/chem.200800065. Farkaš, P., & Bystrický, S. (2008). Hydrolysis of the terminal dimethylacetal moiety on the spacers bound to carboxy groups containing glucans. Carbohydrate Polymers, 74, 133– 136. DOI: 10.1016/j.carbpol.2008.01.005. Farkaš, P., & Bystický, S. (2007). Efficient activation of carboxyl polysaccharides for the preparation of conjugates. Carbohydrate Polymers, 68, 187–190. DOI: 10.1016/j.carbpol.2006. 07.013. García, A., Hernández, K., Chico, B., García, D., Villalonga, M. L., & Villalonga, R. (2009). Preparation of thermostable

A

ut

ho

rc op y

Ac ACVA

References Albericio, F. (2004). Developments in peptide and amide synthesis. Current Opinion in Chemical Biology, 8, 211–221. DOI: 10.1016/j.cbpa.2004.03.002. Amir-Kroll, H., Nussbaum, G., & Cohen, I. R. (2003). Proteins and their derived peptides as carriers in a conjugate vaccine for Streptococcus pneumoniae: Self-heat shock protein 60 and tetanus toxoid. The Journal of Immunology, 170, 6165–6171. Anderson, P., Pichichero, M. E., & Insel, R. A. (1985). Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or CRM197. The Journal of Clinical Investigation, 76, 52–59. DOI: 10.1172/JCI111976. Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang, P. V., Miller, I, A., Lo, A., Codelli, J. A., & Bertozzi, C. R. (2007). Copper-free click chemistry for dynamic in vivo imaging. Proceedings of the National Academy of Sciences,

693

694


Lees, A., Sen, G., & Acosta, A. L. (2006). Versatile and efficient synthesis of protein–polysaccharide conjugate vaccines using aminooxy reagents and oxime chemistry. Vaccine, 24, 716– 729. DOI: 10.1016/j.vaccine.2005.08.096. Leung, C., Chibba, A., Gómez-Biagi, R. F., & Nitz, M. (2009). Efficient synthesis and protein conjugation of β-(1→6)-D-Nacetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin. Carbohydrate Research, 344, 570–575. DOI: 10.1016/j.carres.2008.12.021. Li, C.-J. (2005). Organic reactions in aqueous media with a focus on carbon–carbon bond formations: A decade update. Chemical Reviews, 105, 3095–3165. DOI: 10.1021/cr030009u. Lin, F. L., Hoyt, H. M., van Halbeek, H., Bergman, R. G., & Bertozzi, C. R. (2005). Mechanistic investigation of the Staudinger ligation. Journal of the American Chemical Society, 127, 2686–2695. DOI: 10.1021/ja044461m. Lutz, J.-F., & B¨ orner, H. G. (2008). Modern trends in polymer bioconjugates design. Progress in Polymer Science, 33, 1–39. DOI: 10.1016/j.progpolymsci.2007.07.005. Lutz, J.-F., & Zarafshani, Z. (2008). Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide–alkyne “click” chemistry. Advanced Drug Delivery Reviews, 60, 958–970. DOI: 10.1016/j.addr.2008.02. 004. Mieszala, M., Kogan, G., & Jennings, H. J. (2003). Conjugation of meningococcal lipooligosaccharides through their lipid A terminus conserves their inner epitopes and results in conjugate vaccines having improved immunological properties. Carbohydrate Research, 338, 167–175. DOI: 10.1016/S00086215(02)00395-6. Montalbetti, C. A. G. N., & Falque, V. (2005). Amide bond formation and peptide coupling. Tetrahedron, 61, 10827–10852. DOI: 10.1016/j.tet.2005.08.031. Pavliakova, D., Chu, C., Bystricky, S., Tolson, N. W., Shiloach, J., Kaufman, J. B., Bryla, D. A., Robbins, J. B., & Schneerson, R. (1999). Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri Type 2a O-specific polysaccharide-protein conjugates in mice. Infection and Immunity, 67, 5526–5529. Pawlowski, A., Källenius, G., & Svenson, S. B. (2000). Preparation of pneumococcal capsular polysaccharide-protein conjugate vaccines utilizing new fragmentation and conjugation technologies. Vaccine, 18, 1873–1885. DOI: 10.1016/S0264410X(99)00336-9. Pawlowski, A., Källenius, G., & Svenson, S. B. (1999). A new method of non-cross-linking conjugation of polysaccharides to proteins via thioether bonds for the preparation of saccharide–protein conjugate vaccines. Vaccine, 17, 1474– 1483. DOI: 10.1016/S0264-410X(98)00385-5. Pozsgay, V., & Kubler-Kielb, J. (2008). Conjugation methods toward synthetic vaccines. In R. Roy (Ed.), Carbohydrate based vaccines (pp. 36–70). Washington, DC, USA: American Chemical Society. Pozsgay, V., Vieira, N. E., & Yergey, A. (2002). A method for bioconjugation of carbohydrates using Diels–Alder cycloaddition. Organic Letters, 4, 3191–3194. DOI: 10.1021/ol026179v. Rideout, D. C., & Breslow, R. (1980). Hydrophobic acceleration of Diels–Alder reactions. Journal of the American Chemical Society, 102, 7816–7817. DOI: 10.1021/ja00546a048. Rodionov, V. O., Presolski, S. I., Gardinier, S., Lim, Y.H., & Finn, M. G. (2007). Benzimidazole and related ligands for Cu-catalyzed azide−alkyne cycloaddition. Journal of the American Chemical Society, 129, 12696–12704. DOI: 10.1021/ja072678l. Rogers, L. K., Leinweber, B. L., & Smith, C. V. (2006). Detection of reversible protein thiol modifications in tissues. Analytical Biochemistry, 358, 171–184. DOI: 10.1016/j.ab.2006. 08.020.

A

ut

ho

rc op y

trypsin–polysaccharide neoglycoenzymes through Ugi multicomponent reaction. Journal of Molecular Catalysis B: Enzymatic, 59, 126–130. DOI: 10.1016/j.molcatb.2009.02.001. Grabarek, Z., & Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Analytical Biochemistry, 185, 131–135. DOI: 10.1016/0003-2697(90)90267-D. Grandjean, C., Boutonnier, A., Dassy, B., Fournier, J.-M., & Mulard, L. A. (2009). Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba. Glycoconjugate Journal, 26, 41–55. DOI: 10.1007/s10719-008-9160-6. Grandjean, C., Boutonnier, A., Guerreiro, C., Fournier, J.-M., & Mulard, L. A. (2005). On the preparation of carbohydrateprotein conjugates using the traceless Staudinger ligation. Journal of the Organic Chemistry, 70, 7123–7132. DOI: 10.1021/jo0505472. Griesbaum, K. (1970). Problems and possibilities of the freeradical addition of thiols to unsaturated compounds. Angewandte Chemie International Edition, 9, 273–287. DOI: 10.1002/anie.197002731. Gurd, F. R. N. (1967). Carboxymethylation. Methods in Enzymology, 11, 532–541. DOI: 10.1016/S0076-6879(67)11064-1. Hermanson, G. T. (1996). Bioconjugate techniques. San Diego, CA, USA: Academic Press, Inc. Hou, S.-J., Saksena, R., & Kováč, P. (2008). Preparation of glycoconjugates by dialkyl squarate chemistry revisited. Carbohydrate Research, 343, 196–210. DOI: 10.1016/j.carres. 2007.10.015. Hoyle, C. E., & Bowman, C. N. (2010). Thiol–ene click chemistry. Angewandte Chemie International Edition, 49, 1540– 1573. DOI: 10.1002/anie.200903924. Izumi, M., Okumura, S., Yuasa, H., & Hashimoto, H. (2003). Mannose-BSA conjugates: Comparison between commercially available linkers in reactivity and bioactivity. Journal of Carbohydrate Chemistry, 22, 317–329. DOI: 10.1081/CAR120023475. Johnson, E. C. B., & Kent, S. B. H. (2006). Insights into the mechanism and catalysis of the native chemical ligation reaction. Journal of the American Chemical Society, 128, 6640– 6646. DOI: 10.1021/ja058344i. Jonkheijm, P., Weinrich, D., K¨ ohn, M., Engelkamp, H., Christianen, P. C. M., Kuhlmann, J., Maan, J. C., N¨ usse, D., Schroeder, H., Wacker, R., Breinbauer, R., Niemeyer, C. M., & Waldmann, H. (2008). Photochemical surface patterning by the thiol-ene reaction. Angewandte Chemie International Edition, 47, 4421–4424. DOI: 10.1002/anie.200800101. Kiick, K. L., Saxon, E., Tirrell, D. A., & Bertozzi, C. R. (2002). Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proceedings of the National Academy of Sciences, 99, 19–24. DOI: 10.1073/pnas.012583299. Kohn, J., & Wilchek, M. (1983). 1-Cyano-4-dimethylamino pyridinium tetrafluoroborate as a cyanylating agent for the covalent attachment of ligand to polysaccharide resins. FEBS Letters, 154, 209–210. DOI: 10.1016/0014-5793(83)80905-3. K¨ ohn, M., & Breinbauer, R. (2004). The Staudinger ligation – a gift to chemical biology. Angewandte Chemie International Edition, 43, 3106–3116. DOI: 10.1002/anie.200401744. Kubler-Kielb, J., Liu, T.-Y., Mocca, C., Majadly, F., Robbins, J. B., & Schneerson, R. (2006). Additional conjugation methods and immunogenicity of Bacillus anthracis poly-γ-D-glutamic acid-protein conjugates. Infection and Immunity, 74, 4744– 4749. DOI: 10.1128/IAI.00315-06. Kubler-Kielb, J., & Pozsgay, V. (2005). A new method for conjugation of carbohydrates to proteins using an aminooxy-thiol heterobifunctional linker. Journal of the Organic Chemistry, 70, 6987–6990. DOI: 10.1021/jo050934b.


Soellner, M. B., Nilsson, B. L., & Raine, R. T. (2006). Reaction mechanism and kinetics of the traceless Staudinger ligation. Journal of the American Chemical Society, 128, 8820–8828. DOI: 10.1021/ja060484k. ¨ Staudinger, H., & Meyer, J. (1919). Uber neue organische Phosphorverbindungen III. Phosphinmethylenderivate und phosphinimine. Helvetica Chimimica Acta, 2, 635–646. DOI: 10.1002/hlca.19190020164. Tietze, L. F., Schr¨ oter, C., Gabius, S., Brinck, U., GoerlachGraw, A., & Gabius, H.-J. (1991). Conjugation of paminophenyl glycosides with squaric acid diester to a carrier protein and the use of the neoglycoprotein in the histochemical detection of lectins. Bioconjugate Chemistry, 2, 148–153. DOI: 10.1021/bc00009a003. Wang, J. Y., Chang, A. H. C., Guttormsen, H.-K., Rosas, A. L., & Kaspe, D. L. (2002). Construction of designer glycoconjugate vaccines with size-specific oligosaccharide antigens and site-controlled coupling. Vaccine, 21, 1112–1117. DOI: 10.1016/S0264-410X(02)00625-4. Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., & Finn, M. G. (2003). Bioconjugation by copper(I)-catalyzed azide-alkine [3 + 2] cycloaddition. Journal of the American Chemical Society, 125, 3192–3193. DOI: 10.1021/ja021381e. Xin, H., Dziadek, S., Bundle, D. R., & Cutler, J. E. (2008). Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis. Proceedings of the National Academy of Sciences, 105, 13526–13531. DOI: 10.1073/pnas.0803195105. Xue, J., Pan, Y., & Guo, Z. (2002). Neoglycoprotein cancer vaccines: synthesis of an azido derivative of GM3 and its efficient coupling to proteins through a new linker. Tetrahedron Leters, 43, 1599–1602. DOI: 10.1016/S0040-4039(02)00071-0. Zhang, J., Yergey, A., Kowalak, J., & Kováč, P. (1998). Linking carbohydrates to proteins using N-(2,2-dimethoxyethyl)6-hydroxy hexanamide. Tetrahedron, 54, 11783–11792. DOI: 10.1016/S0040-4020(98)83039-1.

A

ut

ho

rc op y

Rostovtsev, V. V., Green, L. G., Fokin, V. V., & Sharpless, K. B. (2002). A stepwise Huisgen cycloaddition process: Copper(I)catalyzed regioselective ligation of azides and terminal alkynes. Angewandte Chemie International Edition, 41, 2596–2599.DOI: 10.1002/1521-3773(20020715)41:143.0.CO;2-4. Sanki, A. K., Talan, R. S., & Sucheck, S. J. (2009). Synthesis of small glycopeptides by decarboxylative condensation and insight into the reaction mechanism. The Journal of Organic Chemisty, 74, 1886–1896. DOI: 10.1021/jo802278w. Saxon, E., & Bertozzi, C. R. (2000). Cell surface engineering by a modified Staudinger reaction. Science, 287, 2007–2010. DOI: 10.1126/science.287.5460.2007. Schlottmann, S. A., Jain, N., Chirmule, N., & Esser, M. T. (2006). A novel chemistry for conjugating pneumococcal polysaccharides to Luminex microspheres. Journal of Immunological Methods, 309, 75–85. DOI: 10.1016/j.jim.2005. 11.019. Shafer, D. E., Toll, B., Schuman, R. F., Nelson, B. L., Mond, J. J., & Lees, A. (2000). Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides. Vaccine, 18, 1273–1281. DOI: 10.1016/S0264-410X(99)00370-9. Singh, R., & Whitesides, G. M. (1991). A reagent for reduction of disulfide bonds in proteins that reduces disulfide bonds faster than does dithiothreitol. Journal of Organic Chemistry, 56, 2332–2337. DOI: 10.1021/jo00007a018. Sletten, E. M., & Bertozzi, C. R. (2009). Bioorthogonal chemistry: Fishing for selectivity in a sea of functionality. Angewandte Chemie International Edition, 48, 6974–6998. DOI: 10.1002/anie.200900942. Sletten, E. M., & Bertozzi, C. R. (2008). A hydrophilic azacyclooctyne for Cu-free click chemistry. Organic Letters, 10, 3097–3099. DOI: 10.1021/ol801141k.

695