Multicomponent Reactions in Ligation and Bioconjugation Chemistry

Article Cite This: Acc. Chem. Res. 2018, 51, 1475−1486

pubs.acs.org/accounts

Multicomponent Reactions in Ligation and Bioconjugation Chemistry Leslie Reguera,† Yanira Méndez,† Ana R. Humpierre,† Oscar Valdés,‡ and Daniel G. Rivera*,† †

Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba Vicerrectoría de Investigación y Postgrado, Universidad Católica del Maule, Talca 3460000, Chile

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CONSPECTUS: Multicomponent reactions (MCRs) encompass an exciting class of chemical transformations that have proven success in almost all fields of synthetic organic chemistry. These convergent procedures incorporate three or more reactants into a final product in one pot, thus combining high levels of complexity and diversity generation with low synthetic cost. Striking applications of these processes are found in heterocycle, peptidomimetic, and natural product syntheses. However, their potential in the preparation of large macro- and biomolecular constructs has been realized just recently. This Account describes the most relevant results of our group in the utilization of MCRs for ligation/conjugation of biomolecules along with significant contributions from other laboratories that validate the utility of this special class of bioconjugation process. Thus, MCRs have proven to be efficient in the ligation of lipids to peptides and oligosaccharides as well as the ligation of steroids, carbohydrates, and fluorescent and affinity tags to peptides and proteins. In the field of glycolipids, we highlight the power of isocyanide-based MCRs with the one-pot double lipidation of glycan fragments functionalized as either the carboxylic acid or amine. In peptide chemistry, the versatility of the multicomponent ligation strategy is demonstrated in both solution-phase lipidation protocols and solid-phase procedures enabling the simultaneous lipidation and biotinylation of peptides. In addition, we show that MCRs are powerful methods for synchronized lipidation/labeling and macrocyclization of peptides, thus accomplishing in one step what usually requires long sequences. In the realm of protein bioconjugation, MCRs have also proven to be effective in labeling, site-selective modification, immobilization, and glycoconjugation processes. For example, we illustrate a successful application of multicomponent polysaccharide−protein conjugation with the preparation of multivalent glycoconjugate vaccine candidates by the ligation of two antigenic capsular polysaccharides of a pathogenic bacterium to carrier proteins. By highlighting the ability to join several biomolecules in only one synthetic operation, we hope to encourage the biomolecular chemistry community to apply this powerful chemistry to novel biomedicinal challenges.

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they are for the synthesis of small heterocycles6 and peptidomimetics.15 This Account highlights approaches in which the MCR is directly involved in the ligation/conjugation of biomolecules or biomolecular fragmentssuch as fatty acids, steroids, peptides, oligo- and polysaccharides as well as in the labeling, glycoconjugation, and immobilization of proteins. We do not include approaches wherein the MCR is used to ligate only amino acids and monosaccharides, as they are not typically considered to be conjugation methods and there are several reviews3−7 and books1,16 that already summarize such reports. Most multicomponent ligation/conjugation procedures described in the literature rely on isocyanide-based MCRs (IMCRs).16,17 Among this class of MCRs, the Passerini reaction18 and the Ugi reaction19including the Ugi-azide variant20  have proven to be the most useful in conjugation chemistry (Scheme 1A−C). This is largely due to their high efficiency under mild reaction conditions (e.g., room temperature and noninert atmosphere). Because of the abundance of amine

INTRODUCTION

Multicomponent reactions (MCRs) are convergent processes in which three or more reactants are combined in one pot to render a product incorporating atoms from all of the starting materials.1 Because these processes are universally recognized for their high atom economy2 and diversity and complexitygenerating character,1,3 they have been widely exploited in drug discovery1,4 and natural products synthesis.1,5 Important applications of MCRs are also found in heterocycle6 and carbohydrate7 chemistry as well as in the development of organocatalysts8 and modern peptide cyclization strategies.9,10 However, only recently have MCRs been widely exploited in other equally relevant fields such as polymer synthesis11 and biomolecule conjugation.12−14 Over the last years, our laboratory at the University of Havana has been intensively devoted to the development of MCR-based strategies for the multicomponent conjugation of lipids, oligo/polysaccharides, peptides, and proteins. Gratifyingly, this endeavor, along with relevant reports of other groups, has demonstrated the general concept that MCRs are as powerful for the assembly of large biomolecular constructs as © 2018 American Chemical Society

Received: March 20, 2018 Published: May 25, 2018 1475

DOI: 10.1021/acs.accounts.8b00126 Acc. Chem. Res. 2018, 51, 1475−1486

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Accounts of Chemical Research

Ugi ligation to fatty acids, while the amino component enabled the incorporation of a reactive handle in ceramide mimics 1. These handles were meant to allow for subsequent conjugation to sugars, i.e., either a hydroxyl group for glycosylation22 or an azide or alkyne for Cu-catalyzed azide−alkyne cycloaddition (CuAAC) with a functionalized sugar.24 A significant advance in this field was the development of a multicomponent procedure capable of directly conjugating two lipid tails to an oligosaccharide moiety using the Ugi reaction. Scheme 3 illustrates two successful variants of this endeavor

Scheme 1. MCRs Most Commonly Used in Multicomponent Ligation and Bioconjugation Strategies; All of the Substituents (R1−4) Can Be Biomolecular Fragments

Scheme 3. One-Pot Synthesis of Ugi-Derived Neoglycolipids by Multicomponent Ligation of Two Lipids and One Oligosaccharide

groups in peptides and proteins, a common intermediate in bioconjugation approaches is the imine, which is also ubiquitous in all Ugi-type reactions. As highlighted herein, other MCRs having the imine as intermediate have been employed in multicomponent bioconjugation, as in the cases of the Mannich reaction and the metal-catalyzed coupling of an aldehyde, alkyne, and amine (i.e., A3-coupling;21 Scheme 1D).

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LIPID−LIPID AND LIPID−OLIGOSACCHARIDE CONJUGATION To our knowledge, the first report of the multicomponent ligation of fatty acids to other lipidic or glycan fragments was the one-pot synthesis of Ugi-derived ceramide mimics and neoglycolipids described by Brouard and our group.22 Previously, Tron and co-workers had described the synthesis of lipidic isocyanides for conjugation to polyamines.23 As shown in Scheme 2, our concept was to employ lipidic isocyanidesenvisioned as surrogates of sphingolipidsin the Scheme 2. Multicomponent Ligation of a Fatty Acid, Lipidic Isocyanide, and Amine Having a Reactive Handle (i.e., OH, N3, Alkyne) Suitable for Subsequent Conjugation to Sugars

featuring the use of the carbohydrate as either the carboxylic acid or amino component. For this, the trisaccharide βchacotriosecommonly found in steroid saponinswas synthesized and suitably functionalized with the abovementioned groups. Thus, 2′-aminoethyl β-chacotrioside 3 was efficiently conjugated to fatty acids and lipidic isocyanides to render glycolipids 4, thus reducing the number of steps compared with the previous strategy. Additionally, n-dodecyl βchacotrioside derivative 5 having a carboxylic acid at C-6 of the central glucose unit was ligated to two functionalized lipids to furnish glycolipid 6.22 For our group, the success of these approaches opened several possible routes for the multiple functionalization of biomolecules in only one step.

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OLIGOSACCHARIDE−STEROID CONJUGATION After the realization that oligosaccharides could be efficiently conjugated to lipids by I-MCRs, we sought to extend the 1476


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Scheme 4. Synthesis of Steroid−Carbohydrate Conjugates by Multicomponent Ligation of Oligosaccharides to Steroidal Isocyanides

Scheme 5. Multicomponent Ligation of Pseudopeptides to other Peptides, Sugars, and Lipids

of the classic Ugi reaction with spirostanic steroids and the βchacotrioside functionalized as either the amino or carboxylic acid component led to a small library of analogues of cytotoxic diosgenyl β-chacotrioside for anticancer activity evaluation.25 With the aim of further expanding the applications of such steroidal glycosides, we increased the complexity of the multicomponent conjugation process by ligating not one but two oligosaccharide moieties to a bifunctional steroid. This is

strategy and scope to the one-pot assembly of steroidal glycoconjugates. For this, the initial focus was the synthesis and biological evaluation of MCR-derived analogues of steroidal saponins, which required the previous synthesis of structurally novel steroidal isocyanides. As illustrated in Scheme 4, 3βdiosgenyl isocyanide was conjugated to β-chacotriosyl amine by means of the Ugi-azide reaction with generation of HN3 in situ using TMSN3 in MeOH.25 In addition to conjugate 7, the use 1477


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Scheme 6. Multicomponent Conjugation of Two Peptide Fragments to a Steroid for the Construction of Unique N-Steroidal Cyclopeptide Architectures

Scheme 7. Solid-Phase Ligation of Lipids and Steroids to Resin-Bound Peptides by On-Resin Ugi Reaction

amphiphilic cholane−lactose conjugate include its employment as a surfactant for protein solubilization.

exemplified in Scheme 4 with the conjugation of four lactosyl fragments to a cholic acid-derived diisocyanide by a double Ugi reaction,26 thus furnishing steroidal glycoconjugate 8 bearing multiple lactose units positioned at the α face of the concave cholanic skeleton. Although the yield of this conjugation process was only moderate, it led to the assembly of a complex glycoconjugate construct with the formation of eight new covalent bonds in one pot. Current applications of this

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PEPTIDE−PEPTIDE AND CARBOHYDRATE−PEPTIDE LIGATION Whereas the multicomponent assembly of peptidomimetics by reaction of two amino acids is well-established,4,5,15 the ligation of two oligopeptide fragments by means of I-MCRs has been less frequently exploited. It is worth highlighting the pioneering 1478


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Scheme 8. Solid-Phase Conjugation of Lipids, Biotin, and Steroids to Resin-Bound Peptides by On-Resin Ugi and Ugi-Azide Reactions

work of Wessjohann27 and Gross28 on the Ugi ligation of two (pseudo)peptide fragments to produce more stable yet highly active analogues of medicinally important peptides. Another relevant input was the utilization of a highly diastereoselective Ugi ligation procedure during the synthesis of the hepatitis C drug telaprevir (Incivek) by Ruijter and co-workers.29 In collaboration with Paixão’s group, we expanded the diversity of Ugi peptide ligations by developing a highly stereoselective organocatalytic multicomponent approach for the ligation of either two peptides or a peptide and a sugar.30 As shown in Scheme 5A, the innovation of this method lies in the use of enantiomerically enriched enol−hemiacetals as chiral inputs for the conjugation of isocyanopeptides to carbohydrates and aminopeptides to form peptidic hybrids 9 and 10. Another key report in this field is the Ugi multicomponent ligation of a urea−peptide, a lipidic aldehyde, and a hybrid ribosyl−uridine isocyanide during the synthesis of 11, a potent lipophilic antibacterial analogue of muraymycin, as described by Matsuda and co-workers (Scheme 5B).31

these unique N-steroidal peptides can be further cyclized to furnish cyclopeptide−steroid conjugates of type 13, in which the cyclopeptide moiety can be placed at different positions of either the steroidal skeleton or the side chain, as shown in compounds 14, 15, and 16.35 Very recently, our laboratory started a synthetic program for the synthesis of antimicrobial lipopeptides in which the lipidic tail could be positioned not only at the peptide termini but also as an internal amide36 or side-chain37 substituent. To this end, we developed a solid-phase methodology enabling the on-resin multicomponent conjugation of lipids and steroids to peptides. The solid-phase ligation of two different biomolecules, such as lipids, steroids, or polypeptides, had never been accomplished by MCRs. As depicted in Scheme 7A, the on-resin Ugi reaction allows the direct conjugation of a lipid chain or a steroidal skeleton to a resin-bound peptide, while further amino acid couplings enable the subsequent growth of the peptide sequence.36 Scheme 7B highlights some of the lipopeptides (17, 18, and 20) and a peptide−steroid conjugate (19) obtained by this method. A key feature of these conjugates is that they bear the lipid and steroid moieties located either at the C-terminus or as internal N-substituents, which is not so easy to achieve by other standard methods. After proving the success of the solid-phase multicomponent ligation at different positions of the peptide sequence, we turned to implementation of the double ligation of a resinbound peptide at the N-terminus. As shown in Scheme 8, the method allowed the multicomponent incorporation of two lipids (22) and a lipid and a biotin fragment (24) into a decapeptide in only one step. Alternatively, the Ugi-azide reaction also enabled the introduction of either a lipid (21) or steroidal (23) moiety during the formation of the tetrazole ring. Overall, the easy production of such complex peptide conjugates proves the power of this solid-phase multi-

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PEPTIDE−STEROID AND PEPTIDE−LIPID CONJUGATION The multicomponent conjugation of individual amino acids to steroids was first reported by our group in 2006 as part of a strategy for the synthesis of peptide−steroid conjugates.32 Follow-up applications included the synthesis of steroid−amino acid hybrid macrocycles33 and bis-steroidal conjugates.34 Recently, in cooperation with Wessjohann’s group, we extended this concept to the synthesis of a first-in-class family of peptide−steroid conjugates 12 featuring the peptide backbone with the steroid skeleton as an N-substituent.35 As illustrated in Scheme 6, this was achieved by multicomponent conjugation of a peptide carboxylic acid and an isocyanopeptide (or isocyanoacetate) to a steroidal amine. As shown below, 1479


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Scheme 9. (A) Structures of Natural Cyclic Lipopeptides; (B) Simultaneous Cyclization and Lipidation of Peptides by the Ugi and Passerini Reactions

Scheme 10. Simultaneous Cyclization and Lipidation/Labeling of Peptides by On-Resin Ugi−Smiles Reaction

in Scheme 9B, the method enables the conjugation of either one or two exocyclic lipid tails along with the macrocyclic ring closure, which is only possible because of the multicomponent nature of the macrocyclization step. A key feature of the Ugiderived mycosubtilin analogue 27 is that the exocyclic lipid appendage arising from the isocyanide component appears as a substituent of the resulting tertiary amide instead of at the βposition of the β-amino acid. As surfactin features a lactone macrocyclic ring, we chose the Passerini reaction for the macrolactone formation and the simultaneous incorporation of

component approach and paves the way for the introduction of other equally relevant fragments such as poly(ethylene glycol)s (PEGs), glycans, and fluorescent labels. In collaboration with Wessjohann’s group, we developed an efficient procedure for the synthesis of cyclic lipopeptide analogues of the natural products mycosubtilin (25) and surfactin A (26) (Scheme 9A).38 Our approach to this distinctive class of microbial natural products relied on the utilization of the Ugi and Passerini reactions for the simultaneous cyclization and lipidation of peptides. As shown 1480


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Scheme 11. Multicomponent Protein Bioconjugation to (A) a Polymeric Support and (B) Carbohydrates and a Fluorescent Label by Means of the Ugi Reaction

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the exocyclic lipid tail in the synthesis of analogue 28.38 Alternatively, the Ugi macrocyclization employing a lipidic amine and an isocyanide led to surfactin analogue 29 bearing two exocyclic lipids in only one synthetic operation. These results encompass a new example of the potential of MCRs to produce natural product analogues for screening of their biological and chemical properties. Inspired by the success of the Ugi lipidation procedures, we aimed at expanding the repertoire of MCRs suitable for the multicomponent synthesis of cyclic lipopeptides.39 For this, we chose the Ugi−Smiles reaction,40 an Ugi reaction variant not previously used to modify oligopeptides. This I-MCR comprises the replacement of the carboxylic acid component of the classic Ugi reaction by an o- or p-nitrophenol. As shown in Scheme 10, this multicomponent cycloligation strategy encompasses the solid-phase assembly of 3-nitrotyrosinecontaining peptides followed by on-resin imine formation and Ugi−Smiles macrocyclization with an amino group of either a Lys side chain or the N-terminus. Again, the focus was the simultaneous cyclization of the peptide skeleton and its ligation to a lipidic moiety, thus leading to structurally novel N-arylbridged cyclic lipopeptides (30−32). Not only lipid tails but also a fluorescent label (33) could be incorporated at the N-aryl bridge during this multicomponent cycloligation process. This report39 embodied the first application of an Ugi-type reaction in the solid-phase simultaneous macrocyclization and lipidation/labeling of peptides.

PROTEIN IMMOBILIZATION, LABELING, AND GLYCOCONJUGATION The conjugation of proteins to other (bio)molecules using MCRs is a field of notable prospective use, especially in areas such as protein labeling and PEGylation, the preparation of antibody−drug conjugates, and the development of synthetic conjugate vaccines.14 The ability to incorporate multiple fluorescent tags, PEG chains, or antigenic saccharides into a protein in only one synthetic operation instead of by a stepwise protocol would represent a significant advance for this field. To our knowledge, the first utilization of an MCR in protein bioconjugation was reported by Marek et al.41 with the immobilization of the enzyme glucose oxidase on a polymeric carrier by means of the Ugi reaction. As depicted in Scheme 11A, the glycoenzyme was subjected to a periodate oxidation to generate aldehyde groups in the glycosidic part and then linked to amino-functionalized glycidyl methacrylate polymers using excess acetic acid and cyclohexyl isocyanide to render the polymer-supported enzyme 34. Importantly, the method proved as efficient as other classic immobilization procedures.41 Intriguingly, other attempts to use MCRs in protein bioconjugation remained elusive for many years. While the glycoenzyme immobilization relied on the generation of oxo functionalities on the glycan moiety, two other Ugi-reactive functional groups are already present in a protein, i.e., the carboxylic acid and the amine. In 2000, Ziegler and co-workers reported the preparation of bovine serum album (BSA) and horseradish peroxidase (HRP) bioconjugates through the Ugi reaction using either the carboxylic acid or amino groups at the biomolecule surface (Scheme 11B).13 1481


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bioconjugation strategy for the preparation of thermostable neoglycoenzymes.44 For this, trypsin was conjugated by its amino groups to both sodium alginate and (carboxymethyl)cellulose to render trypsin−polysaccharide glycoconjugates with good protease activity and better thermostability than the native enzyme. Even though the high occurrence of Lys on protein surfaces is beneficial for biomolecule reactivity, it is indeed detrimental for site-selective bioconjugation.45 In an endeavor to provide a selective approach for protein labeling, Francis’ group developed a Mannich-type multicomponent process for protein conjugation at Tyr residues.46 Tyr is much less frequent than Lys, and critically, the reactivity of its phenolic side chain is orthogonal to that of Cys, Lys, Glu, and Asp side chains. As illustrated in Scheme 13, this multicomponent Mannich-type

Several Ugi-derived glycoconjugates were prepared using monosaccharides as amino and isocyano components. Conjugate 35derived from the simultaneous conjugation of β-Dglucosyl isocyanide and rhodamine B to BSAand glycoconjugate 36 are the most relevant ones. These Ugi bioconjugation procedures usually required 2−4 days for completion, which is also common in other bioconjugation techniques. However, because of such long reaction times, Ziegler’s group eventually detected denaturation of the protein when specific isocyanides were used, likely as a result of protein cross-linking.13 A solution to increase the protein reactivity of the amino component and thus reduce the reaction time was recently provided by our group with the use of hydrazideactivated proteins in Ugi reactions.14 In the field of polysaccharide conjugation, Crescenzi and coworkers pioneered the utilization of I-MCRs such as Passerini and Ugi reactions for the production of biocompatible synthetic hydrogels.12 This group carried out the cross-linking of (carboxymethyl)cellulose with diamino and dialdehyde building blocks for the assembly of the hydrophilic networks, while 1(deoxylactit-1-yl)chitosan (amino component) was also crosslinked with tartaric acid in the presence of cyclohexyl isocyanide. Alternatively, the immobilization of enzymes in a polysaccharide network by means of I-MCRs was first reported by Ugi himself in 1991.42 An important progress in this field was provided by Villalonga’s groupin cooperation with ourswith the development of an amperometric enzyme biosensor for hydrogen peroxide.43 As shown in Scheme 12, the Ugi reaction

Scheme 13. Site-Selective Protein Labeling at Tyr Residues by a Multicomponent Mannich-Type Bioconjugation

Scheme 12. Multicomponent Immobilization of Horseradish Peroxidase on a Polysaccharide-Coated Gold Electrode

labeling comprises the mild reaction of the Tyr phenol ring with imines derived from aldehydes and electron-rich anilines carrying a rhodamine tag. Varied proteins having surfaceaccessible Tyr residues, such as chymotrypsinogen A, lysozyme, and RNase, were successfully modified with an anilinecontaining tag and various aldehyde components to render labeled proteins 38.46 To our knowledge, this approach encompassed the first site-selective protein conjugation method based on an MCR, and it was additionally employed for the attachment of aniline-containing polypeptides to proteins.47 As depicted in Scheme 14, another interesting example of site-selective protein modification with MCRs is the report by Rai and co-workers on protein labeling (39) using CuIcatalyzed A3-coupling.48 Interestingly, the use of formaldehyde blocks the N-terminal amino group through imidazolidinone formation with the neighboring backbone amide. Additionally, a careful screening of CuI catalyst ligands and reaction conditions provided an effective and selective method for the multicomponent monolabeling of proteins at only one Lys side chain under physiological conditions. The fact that a single Lys residue is modified selectively in the presence of multiple Lys copies and other nucleophilic amino acid side chains is remarkable. Whereas the structure of the labeling moiety (i.e.,

was effectively employed for the immobilization of HRP on a gold electrode previously coated with sodium alginate. The strategy comprised functionalization of the polysaccharide with thiol groups through periodate oxidation followed by reductive amination with 2-aminoethane-1-thiol. Once the anionic biopolymer was supported on the gold electrode, the Ugi bioconjugation procedureusing the protein as the amino componentled to the electrode-immobilized enzyme (37), which showed full activity after 1 month of storage at 4 °C. Villalonga and co-workers also implemented a similar Ugi 1482


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remained unconjugated even after 48 h of reaction, likely because of inefficient imine formation between the protein and the oxo-functionalized CPs. Since detoxification of the native toxins comprises capping several Lys side chains, the low number and poor accessibility of amino groups might be a reason for the poor conjugation efficiency of the protocol leading to glycoconjugate 40. To solve this problem, we turned to activation of both toxoids by reaction of glutamic and aspartic acid side chains with hydrazine, thus increasing the number of the amino components in the hydrazide-activated toxoids TTa and DTa. Scheme 15B illustrates the Ugi bioconjugation of the oxo-CPs to the hydrazide-activated toxoids to form glycoconjugates of type 41. This latter procedure was completed in only 4 h, proving the efficacy of the hydrazide activation strategy. Similar success was accomplished with the conjugation of the antigen of S. Typhi, namely, polysaccharide Vi. This very large polysaccharide features a repetitive 2-acetamidogalacturonic acid unit, so it provided the carboxylic acid component during the Ugi conjugation to TTa and DTa in the presence of acetone and tert-butyl isocyanide. All of the glycoconjugates of DTa and TTa with the CPs of S. pneumoniae and S. Typhi showed notable antigenicity and elicited good titers of functional specific antibodies, thus showing promise for their use as antibacterial conjugate vaccines. After proving that CPs could be efficiently conjugated to carrier proteins as both oxo and carboxylic acid components, we achieved a synthetic landmark not previously attained by any other known bioconjugation method, i.e., the conjugation of two different polysaccharides to a protein in a single step. As depicted in Scheme 16, this was implemented using 2,2,6,6tetramethylpiperidin-1-oxyl (TEMPO)-oxidized CPs14 as the carboxylic acid component, periodate-oxidized CPs7F as the oxo component, and TTa as the amino component of the Ugi bioconjugation, thus leading to glycoconjugate 42 that incorporates two different polysaccharide antigens into a carrier protein. This bivalent unimolecular glycoconjugate showed dual polysaccharide antigenicity, thus confirming the integrity of the antigenic determinants of both CPs after the multicomponent

Scheme 14. Site-Selective Protein Labeling at Only One Lys Residue by a Multicomponent CuI-Catalyzed A3-Coupling Bioconjugation

phenylacetylene) is rather simple, the method proved highly efficient and reproducible under mild operational conditions with a diverse set of nine proteins, including RNase A, ubiquitin, myoglobin, and cytochrome c, among others. Recently, the cooperation between our laboratory and VérezBencomo’s group at the Finlay Institute of Vaccines in Cuba resulted in the development of an important application of multicomponent protein−polysaccharide conjugation in the field of antibacterial glycoconjugate vaccines.14 The approach comprises utilization of the Ugi reaction for bioconjugation of functionalized capsular polysaccharides (CPs) of Streptococcus pneumoniae (S. pneumoniae) and Salmonella enterica serovar Typhi (S. Typhi) to carrier proteins such as diphtheria and tetanus toxoids (DT and TT, respectively). As depicted in Scheme 15A, three different oxo-functionalized CPs of S. pneumoniae (derived from periodate oxidation) were initially conjugated to DT and TT in excess acetic acid and tert-butyl isocyanide. However, about 40% of the carrier proteins

Scheme 15. Multicomponent Bioconjugation of Oxo-Functionalized CPs of S. pneumoniae to (A) Nonactivated DT and TT and (B) Hydrazide-Activated DT and TT; (C) Repetitive Units of the CPs of S. pneumoniae Serotypes 14, 7F, and 9V Used as Oxo Components after Periodate Oxidation

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Scheme 16. Multicomponent Bioconjugation of Two Capsular Polysaccharides of S. pneumoniae to Hydrazide-Activated TT for the Construction of Multivalent Glycoconjugate Vaccines

Biographies

bioconjugation. Similarly, it proved double immunogenicity, i.e., immunogenic behavior in terms of functional immunoglobulin G titers equivalent to those of its monovalent analogues. This result validated the potential of the multicomponent bioconjugation methodology for the development of multivalent vaccine candidates.

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Leslie Reguera graduated in Chemistry with Honors from the University of Havana in 2002. She conducted her doctoral work at both the Universidad Nacional Autónoma de México and the University of Havana, where she did the Ph.D. defense in 2009 with the “Best Ph.D. Thesis of the Year” Award. She has been a Visiting Scientist at the National Polytechnic Institute of Mexico and the University of Leipzig in Germany. Currently she is an Associate Professor of Physical Chemistry at the University of Havana. Her research interests focus on protein immobilization and the development of coordination polymers for catalysis and gas adsorption.

SUMMARY AND OUTLOOK

We have shown that MCRs are versatile synthetic tools for the covalent modification and conjugation of medium-sized and large biomolecules. A variety of examples demonstrate the relevance of MCRs in fields such as antimicrobial peptides, biosensor technology, and antibacterial conjugate vaccines. In this regard, MCRs such as Ugi-type reactions have proven to be effective in carbohydrate and peptide lipidation strategies, in protein glycoconjugation approaches, and in the construction of structurally unique steroidal cyclopeptides and glycosides. MCRs are also well-suited for the development of protein siteselective modification methods, as proven with Mannich-type and metal-catalyzed A3-coupling reactions. However, there is still much to explore for extending the scope of MCRs to other important areas such as antibody−drug conjugates, PEGylated proteins, anticancer glycoconjugate vaccines, and antigenic multivalent peptides. We believe that MCRs will emerge as equally powerful techniques in these areas, as the synthetic power of incorporating not one but multiple payloadse.g., cytotoxic compounds, tumor-associated carbohydrate antigens, PEG chains, or peptide fragmentsinto another biomolecule in only one conjugation step is a key asset for bioconjugation chemists.

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Yanira Méndez was born in Sancti Spı ́ritus, Cuba, in 1990. She received her B.Sc. (First Honors) and M.Sc. degrees in Chemistry from the University of Havana in 2013 and 2016, respectively. Currently she is a Ph.D. student with a DAAD binational doctoral project between the groups of Prof. Bernhard Westermann in Germany and Prof. Rivera in Havana. Her research topic focuses on bioconjugation techniques and drug discovery. Ana R. Humpierre was born in Havana in 1994 and graduated in Chemistry with Honors from the University of Havana in 2017, defending the Diploma Thesis with the maximum qualification. She is currently a Ph.D. student at the University of Havana working in Prof. Rivera’s group on the development of bioconjugation techniques for glycoconjugate vaccines. Oscar Valdés studied chemistry at the University of Havana, where he obtained his Master’s degree in 2005. He earned his Ph.D. in 2010 at the same university, working on the development of scaffolds from natural and synthetic polymers for biomedical applications. He undertook postdoctoral studies at the Center for Systems Biotechnology, Fraunhofer Chile Research, and since May 2017 he has been affiliated with the Universidad Católica del Maule in Chile as an Associate Researcher. His research interests are the development of new materials for low-cost filtration technologies and applications of macromolecules and bioconjugates.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +53 78792331. E-mail: [email protected].

Daniel G. Rivera graduated in Chemistry with Honors from the University of Havana in 2002 and completed his Master’s thesis in Organic Chemistry in the same year. He earned the Ph.D. (Suma Cum Lauden) in 2007, working on the development of multicomponent macrocyclization strategies in the group of Prof. L. A. Wessjohann at the Leibniz Institute of Plant Biochemistry (IPB) in Germany. After

ORCID

Daniel G. Rivera: 0000-0002-5538-1555 Notes

The authors declare no competing financial interest. 1484


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Through Multiple Multicomponent Macrocyclizations. Angew. Chem., Int. Ed. 2017, 56, 3501−3505. (b) Ricardo, M. G.; Morales, F. E.; Garay, H.; Reyes, O.; Vasilev, D.; Wessjohann, L. A.; Rivera, D. G. Bidirectional Macrocyclization of Peptides by Double Multicomponent Reactions. Org. Biomol. Chem. 2015, 13, 438−446. (c) Vasco, A. V.; Pérez, C. S.; Morales, F. E.; Garay, H. E.; Vasilev, D.; Gavín, J. A.; Wessjohann, L. A.; Rivera, D. G. Macrocyclization of Peptide Side Chains by the Ugi Reaction: Achieving Peptide Folding and Exocyclic N-Functionalization in One Shot. J. Org. Chem. 2015, 80, 6697−6707. (11) Kreye, O.; Tóth, T.; Meier, M. A. R. Introducing Multicomponent Reactions to Polymer Science: Passerini Reactions of Renewable Monomers. J. Am. Chem. Soc. 2011, 133, 1790−1792 and articles citing this pioneering work. (12) de Nooy, A. E. J.; Masci, G.; Crescenzi, V. Versatile Synthesis of Polysaccharide Hydrogels Using the Passerini and Ugi Multicomponent Condensations. Macromolecules 1999, 32, 1318−1320. (13) Ziegler, T.; Gerling, S.; Lang, M. Preparation of Bioconjugates through an Ugi Reaction. Angew. Chem., Int. Ed. 2000, 39, 2109−2112. (14) Méndez, Y.; Chang, J.; Humpierre, A. R.; Zanuy, A.; Garrido, R.; Vasco, A. V.; Pedroso, J.; Santana, D.; Rodríguez, L. M.; García-Rivera, D.; Valdés, Y.; Vérez-Bencomo, V.; Rivera, D. G. Multicomponent Polysaccharide−Protein Bioconjugation in the Development of Antibacterial Glycoconjugate Vaccine Candidates. Chem. Sci. 2018, 9, 2581−2588. (15) Wessjohann, L. A.; Rhoden, C. R.; Rivera, D. G.; Vercillo, O. E. Cyclic Peptidomimetics and Pseudopeptides from Multicomponent Reactions. In Synthesis of Heterocycles via Multicomponent Reactions I; Orru, R. V. A., Ruijter, E., Eds.; Springer: Berlin, 2010; pp 199−226. (16) Nenajdenko, V. G. Isocyanide Chemistry: Applications in Synthesis and Material Science; Wiley-VCH: Weinheim, Germany, 2012. (17) Dö mling, A.; Ugi, I. Multicomponent Reactions with Isocyanides. Angew. Chem., Int. Ed. 2000, 39, 3168−3210. (18) Banfi, L.; Riva, R. The Passerini Reaction. Org. React. 2005, 65, 1−140. (19) Ugi, I. Versuche mit Isonitrilen. Angew. Chem. 1959, 71, 386. (20) Ugi, I.; Bodesheim, F. Isonitrile, VIII. Umsetzung von Isonitrilen mit Hydrazonen und Stickstoffwasserstoffsaure. Chem. Ber. 1961, 94, 2797−2801. (21) Peshkov, V. A.; Pereshivko, O. P.; van der Eycken, E. V. A Walk around the A3-Coupling. Chem. Soc. Rev. 2012, 41, 3790−3807. (22) Pérez-Labrada, K.; Brouard, I.; Méndez, I.; Rivera, D. G. Multicomponent Synthesis of Ugi-Type Ceramide Analogues and Neoglycolipids from Lipidic Isocyanides. J. Org. Chem. 2012, 77, 4660−4670. (23) Pirali, T.; Callipari, G.; Ercolano, E.; Genazzani, A. A.; Giovenzana, G. B.; Tron, G. C. A Concise Entry into Nonsymmetrical Alkyl Polyamines. Org. Lett. 2008, 10, 4199−4202. (24) Pérez-Labrada, K.; Brouard, I.; Méndez, I.; Pérez, C. S.; Gavín, J. A.; Rivera, D. G. Combined Ugi-4CR/CuAAC Approach to TriazoleBased Neoglycolipids. Eur. J. Org. Chem. 2014, 2014, 3671−3683. (25) Rivera, D. G.; Pérez-Labrada, K.; Lambert, L.; Dörner, S.; Westermann, B.; Wessjohann, L. A. Carbohydrate−Steroid Conjugation by Ugi Reaction: One-Pot Synthesis of Triple Sugar/PseudoPeptide/Spirostane Hybrids. Carbohydr. Res. 2012, 359, 102−110. (26) Rivera, D. G.; León, F.; Concepción, O.; Morales, F. E.; Wessjohann, L. A. A Multiple Multicomponent Approach to Chimeric Peptide−Peptoid Podands. Chem. - Eur. J. 2013, 19, 6417−6428. (27) Pando, O.; Stark, S.; Denkert, A.; Porzel, A.; Preusentanz, R.; Wessjohann, L. A. The Multiple Multicomponent Approach to Natural Product Mimics: Tubugis, N-Substituted Anticancer Peptides with Picomolar Activity. J. Am. Chem. Soc. 2011, 133, 7692−7695. (28) Arabanian, A.; Mohammadnejad, M.; Balalaie, S.; Gross, J. H. Synthesis of Novel Gn-RH Analogues Using Ugi-4MCR. Bioorg. Med. Chem. Lett. 2009, 19, 887−890. (29) Znabet, A.; Polak, M. M.; Janssen, E.; de Kanter, F. J.; Turner, N. J.; Orru, R. V. A.; Ruijter, E. A Highly Efficient Synthesis of Telaprevir by Strategic Use of Biocatalysis and Multicomponent Reactions. Chem. Commun. 2010, 46, 7918−7920.

returning to his alma mater in 2008, he was appointed Research Professor, Head of Bioorganic Chemistry, and in 2015 Director of the Center for Natural Products Research. He has served as a Visiting Researcher at the Federal University of São Carlos in Brazil, and at the IPB as a Fellow of the Alexander von Humboldt Foundation. His research interests focus on the synthesis of natural products, multicomponent and organocatalytic reactions, and the development of novel macrocyclization and bioconjugation strategies. He is the President of the Cuban Society of Chemistry for 2016−2018 and member of the Cuban Academy of Science.

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ACKNOWLEDGMENTS We are sincerely grateful to all of the students and co-workers who shaped the results presented herein, particularly to Professors Ludger A. Wessjohann, Bernhard Westermann, Ignacio Brouard, and Marcio W. Paixão for their continuous and invaluable support to our research. O.V. is grateful to Fondecyt, Chile (Project 11170008), Y.M. to DAAD, Germany, and D.G.R. to the Alexander von Humboldt Foundation.

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REFERENCES

(1) Multicomponent Reactions in Organic Synthesis; Zhu, J., Wang, Q., Wang, M., Eds.; Wiley-VCH: Weinheim, Germany, 2015. (2) Cioc, R. C.; Ruijter, E.; Orru, R. V. Multicomponent Reactions: Advanced Tools for Sustainable Organic Synthesis. Green Chem. 2014, 16, 2958−2975. (3) (a) Brauch, S.; van Berkel, S. S.; Westermann, B. Higher-Order Multicomponent Reactions: beyond Four Reactants. Chem. Soc. Rev. 2013, 42, 4948−4962. (b) Ruijter, E.; Scheffelaar, R.; Orru, R. V. A. Multicomponent Reaction Design in the Quest for Molecular Complexity and Diversity. Angew. Chem., Int. Ed. 2011, 50, 6234− 6246. (4) (a) Slobbe, P.; Ruijter, E.; Orru, R. V. A. Recent Applications of Multicomponent Reactions in Medicinal Chemistry. MedChemComm 2012, 3, 1189−1218. (b) Dömling, A.; Wang, W.; Wang, K. Chemistry and Biology of Multicomponent Reactions. Chem. Rev. 2012, 112, 3083−3135. (5) Toure, B. B.; Hall, D. G. Natural Product Synthesis Using Multicomponent Reaction Strategies. Chem. Rev. 2009, 109, 4439− 4486. (6) (a) Rotstein, B. H.; Zaretsky, S.; Rai, V.; Yudin, A. K. Small Heterocycles in Multicomponent Reactions. Chem. Rev. 2014, 114, 8323−8359. (b) Dömling, A. Recent Developments in Isocyanide Based Multicomponent Reactions in Applied Chemistry. Chem. Rev. 2006, 106, 17−89. (7) Khan, M. M.; Yousuf, R.; Khan, S.; Shafiullah. Recent Advances in Multicomponent Reactions Involving Carbohydrates. RSC Adv. 2015, 5, 57883−57905. (8) (a) Scatena, G. S.; de la Torre, A. F.; Cass, Q. B.; Rivera, D. G.; Paixão, M. W. Multicomponent Approach to Silica-Grafted Peptide Catalysts: A 3 D Continuous-Flow Organocatalytic System with Online Monitoring of Conversion and Stereoselectivity. ChemCatChem 2014, 6, 3208−3214. (b) de la Torre, A. F.; Rivera, D. G.; Ferreira, M. A. B.; Corrêa, A. G.; Paixão, M. W. Multicomponent Combinatorial Development and Conformational Analysis of Prolyl Peptide−Peptoid Hybrid Catalysts: Application in the Direct Asymmetric Michael Addition. J. Org. Chem. 2013, 78, 10221−10232. (9) (a) Zhang, J.; Mulumba, M.; Ong, H.; Lubell, W. D. DiversityOriented Synthesis of Cyclic Azapeptides by A3-macrocyclization Provides High-Affinity CD36-Modulating Peptidomimetics. Angew. Chem., Int. Ed. 2017, 56, 6284−6288. (b) Frost, J. R.; Scully, C. C. G.; Yudin, A. K. Oxadiazole Grafts in Peptide Macrocycles. Nat. Chem. 2016, 8, 1105−1111. (c) White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide Macrocyclization. Nat. Chem. 2011, 3, 509−524. (10) (a) Wessjohann, L. A.; Kreye, O.; Rivera, D. G. One-Pot Assembly of Amino Acid Bridged Hybrid Macromulticyclic Cages 1485


Article

Accounts of Chemical Research (30) Echemendía, R.; de La Torre, A. F.; Monteiro, J. L.; Pila, M.; Corrêa, A. G.; Westermann, B.; Rivera, D. G.; Paixão, M. W. Highly Stereoselective Synthesis of Natural-Product-Like Hybrids by an Organocatalytic/Multicomponent Reaction Sequence. Angew. Chem., Int. Ed. 2015, 54, 7621−7625. (31) Tanino, T.; Ichikawa, S.; Al-Dabbagh, B.; Bouhss, A.; Oyama, H.; Matsuda, A. Synthesis and Biological Evaluation of Muraymycin Analogues Active against Anti-Drug-Resistant Bacteria. ACS Med. Chem. Lett. 2010, 1, 258−262. (32) Rivera, D. G.; Pando, O.; Coll, F. Synthesis of PeptidomimeticSpirostane Hybrids via Ugi Reaction: a Versatile Approach for the Formation of Peptide−Steroid Conjugates. Tetrahedron 2006, 62, 8327−8334. (33) Wessjohann, L. A.; Rivera, D. G.; Coll, F. Synthesis of Steroid− Biaryl Ether Hybrid Macrocycles with High Skeletal and Side Chain Variability by Multiple Multicomponent Macrocyclization Including Bifunctional Building Blocks. J. Org. Chem. 2006, 71, 7521−7526. (34) Pérez-Labrada, K.; Méndez, Y.; Brouard, I.; Rivera, D. G. Multicomponent Ligation of Steroids: Creating Diversity at the Linkage Moiety of Bis-spirostanic Conjugates by Ugi Reactions. ACS Comb. Sci. 2013, 15, 320−330. (35) Rivera, D. G.; Vasco, A. V.; Echemendía, R.; Concepción, O.; Pérez, C. S.; Gavin, J. A.; Wessjohann, L. A. A Multicomponent Conjugation Strategy to Unique N-Steroidal Peptides: First Evidence of the Steroidal Nucleus as a β-Turn Inducer in Acyclic Peptides. Chem. - Eur. J. 2014, 20, 13150−13161. (36) Morales, F. E.; Garay, H. E.; Muñoz, D. F.; Augusto, Y. E.; Otero-Gonzàlez, A. J.; Reyes Acosta, O.; Rivera, D. G. AminocatalysisMediated On-Resin Ugi Reactions: Application in the Solid-Phase Synthesis of N-Substituted and Tetrazolo Lipopeptides and Peptidosteroids. Org. Lett. 2015, 17, 2728−2731. (37) Wessjohann, L. A.; Morejón, M. C.; Ojeda, G. M.; Rhoden, C. R.; Rivera, D. G. Applications of Convertible Isonitriles in the Ligation and Macrocyclization of Multicomponent Reaction-Derived Peptides and Depsipeptides. J. Org. Chem. 2016, 81, 6535−6545. (38) Morejón, M. C.; Laub, A.; Kaluđerović, G. N.; Puentes, A. R.; Hmedat, A. N.; Otero-González, A. J.; Rivera, D. G.; Wessjohann, L. A. A multicomponent Macrocyclization Strategy to Natural Product-Like Cyclic Lipopeptides: Synthesis and Anticancer Evaluation of Surfactin and Mycosubtilin Analogues. Org. Biomol. Chem. 2017, 15, 3628− 3637. (39) Morejón, M. C.; Laub, A.; Westermann, B.; Rivera, D. G.; Wessjohann, L. A. Solution-and Solid-Phase Macrocyclization of Peptides by the Ugi−Smiles Multicomponent Reaction: Synthesis of N-Aryl-Bridged Cyclic Lipopeptides. Org. Lett. 2016, 18, 4096−4099. (40) El Kaim, L.; Grimaud, L.; Oble, J. Phenol Ugi−Smiles Systems: Strategies for the Multicomponent N-Arylation of Primary Amines with Isocyanides, Aldehydes, and Phenols. Angew. Chem., Int. Ed. 2005, 44, 7961−7964. (41) Marek, M.; Jary, J.; Valentová, O.; Vodrázǩ a, Z. Immobilization of Glycoenzymes by Means of their Glycosidic Components. Biotechnol. Lett. 1983, 5, 653−658. (42) König, S.; Ugi, I. Vernetzung wäßriger Alginsäure mittels der Vierkomponenten-Kondensation unter Einschluß-Immobilisierung von Enzymen. Z. Naturforsch. B 1991, 46, 1261−1266. (43) Camacho, C.; Matías, J. C.; García, D.; Simpson, B. K.; Villalonga, R. Amperometric Enzyme Biosensor for Hydrogen Peroxide via Ugi Multicomponent Reaction. Electrochem. Commun. 2007, 9, 1655−1660. (44) García, A.; Hernández, K.; Chico, B.; García, D.; Villalonga, M. L.; Villalonga, R. Preparation of Thermostable Trypsin−Polysaccharide Neoglycoenzymes through Ugi Multicomponent Reaction. J. Mol. Catal. B: Enzym. 2009, 59, 126−130. (45) Krall, N.; Da Cruz, F. P.; Boutureira, O.; Bernardes, G. J. SiteSelective Protein-Modification Chemistry for Basic Biology and Drug Development. Nat. Chem. 2016, 8, 103−113. (46) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126, 15942−15943.

(47) Romanini, D. W.; Francis, M. B. Attachment of Peptide Building Blocks to Proteins through Tyrosine Bioconjugation. Bioconjugate Chem. 2008, 19, 153−157. (48) Chilamari, M.; Purushottam, L.; Rai, V. Site-Selective Labeling of Native Proteins by a Multicomponent Approach. Chem. - Eur. J. 2017, 23, 3819−3823.

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