Conjugation Methods in Synthetic Glycoconjugate Vaccines

In the past two to three decades, synthetic glycoconjugate vaccines have shown great potential in the prevention of severe infections and protection of high-risk populations. Conjugation of synthetic oligosaccharide haptens to carrier proteins is the key step for the vaccine preparation. In this review, the conjugation methods currently used in the synthesis of glycoconjugate vaccines from synthetic/homogeneous oligosaccharide haptens are summarized with the focus on the reaction conditions (pH and sugar/protein ratio) and performance. This information can help researchers choose the appropriate conjugation methods. Further research directions toward site-specific conjugations and fully homogeneous glycoconjugate vaccines are also discussed.


Introduction
The development and wide application of antibiotics and antimicrobial vaccines significantly change our status in the endless race with infectious diseases.Due to the emerging antibiotic resistances and the slow pace of discovering new antibiotics, the contribution from antibacterial vaccines to human health and public health system will continuously increase in the foreseeable future. [1]All bacterial pathogens are covered with thick layers of polysaccharides (e. g., lipopolysaccharides, capsular polysaccharides, cell wall polysaccharides, teichoic acids) with diverse structures very different from mammalian cells. [2]This becomes the theoretical basis for carbohydrate-based vaccines that use bacteria/fungi-specific carbohydrates as antigens to immunize people and generate protective antibodies.However, the generally low immunogenicity of the microbial polysaccharides elicit only the short-term immune response without memory which makes polysaccharide vaccine less effective, [3] especially to infants under 2 years of age. [4]Based on the early discoveries that conjugating polysaccharides to bacterial proteins or detoxified versions elicited enhanced immune response than polysaccharides themselves, [5] the development of glycoconjugate vaccines become the current approach and has achieved great success in the past four decades. [6]As leading examples, the application and evolution of multivalent Streptococcus pneumoniae vaccines significantly reduce the severe and lethal infection cases especially in young children and elder people. [7]In these onmarket glycoconjugate vaccines, tetanus toxoid (TT), cross reacting material 197 (CRM197), diphtheria toxin (DT), and protein D (glycerophosphodiester phosphodiesterase from Haemophilus influenzae) are applied as carrier proteins in the conjugation with purified partially degraded S. pneumoniae capsular polysaccharides (CPSs) to cover most virulent serotypes.
The generally accepted working mechanism of glycoconjugate vaccines is shown in Figure 1.Currently, two models have been proposed. [8]In the classic model, the carbohydrate hapten on the surface of carrier proteins binds to the B cell receptor (BCR) and determines the specificity of the generated antibodies (mainly IgM).Meanwhile, the glycoconjugate-BCR complex is endocytosed followed by the degradation of the carrier protein.The short peptides formed are displayed on the B cell surface in the peptide-MHC II complex form to stimulate CD4 + T cell via interaction with T cell receptor (TCR), together with the interactions between other costimulatory molecules.The activated T cell releases cytokines to stimulate B cell maturation to memory cells and induce IgM-to-IgG switching.In the novel model, the glycopeptide generated from the degradation of conjugate vaccine can also be displayed in MHC II dependent manner.6c] In most of the clinically used glycoconjugate vaccines, the carbohydrate haptens are heterogeneous polysaccharides obtained from pathogenic bacteria via controlled degradation.Though quite successful in clinical practice, the difficulty in handling infective pathogens and risk from bacterial toxin contaminations can raise safety issues.6c,9] Thus, the synthetic homogeneous oligosaccharides become attractive hapten can-didates for glycoconjugate vaccine development, especially in this era that complex oligosaccharides are accessible from the efficient chemical and chemoenzymatic synthesis.The successful development and approval of Quimi-Hib, a glycoconjugate vaccine against Haemophilus influenzae b based on synthetic oligomeric polyribosylribitol phosphate (sPRP), demonstrated the great potential of synthetic oligosaccharide haptens. [10]ue to the importance of glycoconjugates in chemical biology and vaccine development, some excellent reviews on the glycoconjugation methods have been published. [11]Generally, the structure of synthetic glycoconjugate vaccine is comprised of synthetic oligosaccharide hapten, linker, and carrier protein (Figure 2).6c] Unlike polysaccharide haptens where multiple modification sites are commonly introduced to the polysaccharide chain (e. g., aldehyde groups via NaIO 4 oxidation of vicinal diol and OÀ CN groups via BrCN treatment), synthetic oligosaccharide haptens normally contain single modifiable groups at the reducing end that are installed during the synthesis to facilitate the conjugation.Conjugation methods can be classified into two types.For type 1 conjugations, the modified haptens are conjugated to unmodified carrier proteins via selective reaction with protein side chains (mainly Lys).On the contrary, for type 2 conjugations, both the haptens and carrier proteins need to be modified to install functional group pairs to facilitate conjugation, and Lys residue is still the most used one in the carrier protein modification step.Details of both types of conjugation methods and representative examples will be discussed in the following parts of this review.In this review, we summarized the conjugation methods used in the synthesis of glycoconjugate vaccines from synthetic/homogeneous oligosaccharides with the focus on the reaction conditions (pH and sugar/protein ratio) and performance.We mainly focused on the antibacterial vaccines published after 2000.As another important aspect of glycoconjugate vaccine research, the carbohydrate-based antitumor vaccines [12] is out of the scope of this review, while limited examples are discussed in some cases.The examples will be classified based on the conjugation methods they used.Most of the examples we collected could not be detailly discussed and are summarized in Table 1.We apologize for the contributions which we failed to include.This review could provide useful information to researchers working in the field of synthetic/ semisynthetic glycoconjugate vaccine development, protein modification and bioconjugation.

Disuccinimidyl Ester Conjugation (Type 1)
As an old type of bifunctional reagent, disuccinimidyl (diNHS) esters are used as the crosslinking method of proteins. [13]The stability of the NHS ester moieties in aqueous buffer (moderate but good enough for fast transformations) and good selectivity for the amide formation make them ideal reagents to facilitate the conjugation of amine-modified carbohydrates to carrier proteins.In 1992, Costantino et al. reported the first application of disuccinimidyl adipate 1 in the preparation of glycoconjugate vaccines against Meningococcus A and C, using the isolated capsular polysaccharides. [14]To facilitate amide linkage formation, the amino group was installed via reductive amination at the reducing end of the partially degraded polysaccharide.In 2002, Panza et al. reported the first example using 1 to prepare anticancer glycoconjugate vaccine candidate 3 from synthetic Globo-H antigen derived tetrasaccharide hapten (Figure 3). [15]In their protocol, 10 equiv. of 1 was used in step 1 to ensure aminolysis of one NHS ester, and modified sugar well soluble in water was isolated from the insoluble excess 1 by filtration after removing dioxane by lyophilization.In step 2, the modified sugar was subjected to the conjugation with CRM197 in phosphate buffer (pH 7.4) to give vaccine candidate 3. Using 34 equiv. of sugar led to the installation of 11 haptens on average.Following a similar protocol, up to 248 haptens were installed on KLH using 1820 equiv. of sugar.
As a robust amine-bridging conjugation method, the disuccinimidyl ester conjugation has been applied to the   construction of a series of conjugate vaccines (Figure 3).In 2013, Anish and Seeberger et al. developed an anti-C.difficile vaccine 4 based on the pentasaccharide derived from the surface glycan PSÀ I. [16] In this work, the solvent of step 1 was changed to DMSO with Et 3 N added as base, and the excess reagent 1 was removed via precipitation by adding sodium phosphate buffer and repeated extraction with chloroform.In step 2, 200 equiv. of pentasaccharide derivative was used to achieve 9.6 haptens installed.In 2020, an anti-S.pneumoniae vaccine 5 based on merged 19A/19F hapten was reported by Seeberger et al. [17] Two trisaccharides derived from serotype 19A and 19F (with different Glc-Rha linkages) were linked via phosphate group and the merged hapten (86 equiv.) was conjugated to CRM197 at pH 7.0 to give vaccine candidate with loading 5. To tackle the in vivo stability concern of the oligosaccharide haptens, the carbasugar mimetic which was resistant to glycosidase was used in the anti-N.meningitidis serogroup A glycoconjugate vaccines 6. [18] The synthetic octasaccharide mimetic (50 equiv.)with amino group introduced via phosphate linkage was conjugated to CRM197 at pH 7.2 to achieve loading 10.4.Apart from antibacterial vaccines, the anti-Leishmania vaccine 7 against parasite infection was reported in 2013. [19]The tetrasaccharide hapten derived from the cell surface lipophosphoglycans (LPGs) was conjugated to CRM197 with loading 8-10.More examples using diNHS ester conjugation were summarized in Table 1 (part A).

Di-p-nitrophenyl Ester Conjugation (Type 1)
The di-p-nitrophenyl (diPNP) ester conjugation, commonly using di-p-nitrophenyl adipate reagent 8, was first introduced to glycoconjugate synthesis by Bundle et al. in 2004.Compared to diNHS ester conjugation, the improved stability of PNP ester group allowed the HPLC purification of the sugar derived half ester intermediate. [43]In their model study, the half esters obtained via amidation of monosaccharides/disaccharides derived amines in anhydrous DMF was isolated in 65-82 % yields.
In the subsequent conjugation study at pH 7.5, lactose was installed onto BSA with loading 5, 9, and 16 using 10, 20 and 30 equiv. of half ester respectively.Like diNHS ester conjugation, the diPNP ester conjugation also gained wide applications in synthetic antibacterial glycoconjugate vaccine studies (Figure 4).In 2017, Pereira and Seeberger et al. reported the preparation of anti-S.pneumoniae serotype 5 vaccine. [44]In their protocol, the pentasaccharide 9 derived from the capsular polysaccharide (CPS) of S. pneumoniae ST5 was modified with 10 equiv. of diPNP adipate 8 in the mixture of DMSO and pyridine in the presence of Et 3 N.The half ester generated in step 1 was isolated from the excess 8 by lyophilization and repeated trituration (with chloroform and CH 2 Cl 2 ).In step 2, 58 equiv. of half ester was conjugated to CRM197 at pH 8.0 to give vaccine candidate 10 with loading 12.
Since the emergence of carbapenem-resistant Klebsiella pneumoniae (CR-Kp), the development of anti-CR-Kp vaccine becomes an appealing strategy alternative to discovering new antibiotics to tackle this life-threatening infection.In 2017, Seeberger and Pereira et al. reported a conjugate vaccine 11 based on the complex hexasaccharide hapten derived from the CPS of a CR-Kp strain isolated from a serious hospital outbreak in 2011. [45]The diPNP conjugation gave the CRM197 glycoconjugate with loading 7.5 from 40 equiv. of half ester.In a similar case, the vaccine candidate 12 based on hexasaccharide hapten derived from S. pneumoniae serotype 2 was prepared. [46]The hapten loading 7 was achieved by using 33 equiv. of half ester.
As the analogue of reagent 1, the direct comparison of reagent 8 with 1 was also reported. [47,48]In the construction of anti-S.pneumoniae serotype 2 vaccine 13, [47] when reagent 1 was used, 162 equiv. of tetrasaccharide was transformed into half ester and conjugated to CRM197 at pH 7.4 with loading 9.6.On the contrary, 38.5 equiv. of the half ester obtained via treating tetrasaccharide with reagent 8 was conjugated at pH 8.0 to give conjugate with loading 8.8.The lower reactivity of 8 allowed the conjugation at higher concentration (0.17 mM vs. 0.014 mM for CRM197; 6.5 mM vs. 2.3 mM for half ester) using less excess synthetic oligosaccharides to give comparable hapten loading.However, in the case of anti-C.difficile vaccine, the less reactive reagent 8 gave much lower hapten loading than reagent 1. [48] Thus, both reagents are worth testing for optimizing the conjugation protocol.More examples using diPNP ester conjugation are summarized in Table 1 (part B). [49][50][51][52][53][54]

Squaramide Conjugation (Type 1)
The application of squaric acid diethyl ester 13 as bioconjugation reagent can be dated back to 1991. [55]As an aminebridging reagent with two vicinal reaction sites, the aminolysis of the first ester at neutral pH significantly deactivates the second ester moiety, which needs pH higher than 9.0 for reaction.Thus, reagent 13 (as well as the dimethyl ester analogue) can avoid the formation of undesired symmetric urea-like structure in the sugar derivatization step without being used in large excess amounts.Moreover, the excess amount of half ester species used in the conjugation step can be separated from the conjugates by ultrafiltration and recovered by HPLC. [56]55b] The conjugation protocols and the conjugate characterization were further optimized by Hindsgaul et al. [57] In 1996, Pinto et al. reported the application of 13 in the synthesis of conjugates of group A Streptococcus derived hexasaccharide hapten (Figure 5). [58]In step 1, the hexasaccharide 14 with amino group installed at the reducing end via thiol-ene reaction was treated with 1 equiv. of 13 in MeOH to give half ester in almost pure form without purification, which was simpler than using excess amounts of reagents 1 and 8.In step 2, 26 equiv. of half ester was mixed with BSA in 0.1 M carbonate buffer (pH 10) to give conjugate 15 with hapten loading 16.Though the conjugate 15 was used as immunoadsorbent, the corresponding conjugate to TT protein with hexasaccharide hapten loading 30 was tested as vaccine candidate. [59]s an ideal reagent for glycoconjugate synthesis, 13 has been applied to the development of vaccines against bacteria and fungi.In 2013, Nifantiev et al. reported the synthesis of conjugate 16 from a pentasaccharide hapten derived from the C. albicans cell wall polysaccharide. [60]Conjugate with hapten loading 10 was obtained using 30 equiv. of sugar.In 2014, Bundle et al. developed a conjugate vaccine against Brucella species using a hexasaccharide hapten designed by merging A epitope and M epitope. [61]Using 30 equiv. of hexasaccharide, the conjugate 17 with hapten loading 12-15 was obtained.In 2015, Lombardi et al. reported the conjugation of C-glycoside mimetic of N. meningitidis group A derived hapten (20 equiv.) to CRM197. [62]In the case of trisaccharide hapten, prolonging the conjugation time from 2 h to 6 days led to the significantly increased loading in 18 from 6.5 to 14.2.In 2020, the octasaccharide hapten derived from E. faecalis CPS was conjugated to BSA to generate vaccine candidate 19. [63]As reported by Huebner et al., 30 equiv. of sugar was used to give hapten loading 18 after reaction for 7 days.6][67][68][69] Reductive Amination (Type 1) As mentioned above, reductive amination is commonly used in the polysaccharide based conjugate vaccines.Since the introduction of aldehyde functionality to synthetic oligosaccharide is not facile, reductive amination is only used in special cases (Figure 6).In 2008, Guo et al. reported the antitumor vaccine based on modified STn antigen. [70]The aldehyde group was generated via ozonolysis of the terminal alkene in sugar 20.After gel chromatography purification, the aldehyde intermediate (10 mg) was subjected to reductive amination with KLH protein (10 mg) under slightly basic condition (pH 7.5-8.0)using NaBH 3 CN as reductant.After 4 days reaction, conjugate 21 with hapten loading 4 % wt (~23 haptens/KLH) was obtained.In a recent example reported by Sauvageau et al., the synthetic pentasaccharide hapten derived from P. aeruginosa Aband polysaccharide was conjugated to HSA. [71a] The sugar with free reducing end (65 equiv.)underwent reductive amination at 55 °C for 72 h to give conjugate 22 with hapten loading 6 (further decreased to 1 when reacted at 37 °C), due to the low   The Michael addition of thiol to maleimide is generally acknowledged as a click-type reaction with high efficiency (high reaction rate and yield) and selectivity. [72]The thiol-maleimide conjugation has gained wide applications in protein modifications and material science. [73]Though the stability issue caused by the retro-Michael process has become a concern, [74] the thiol-maleimide conjugation has been applied to the preparation of a plethora of glycoconjugate vaccines, including the only synthetic vaccine under clinical use, Quimi-Hib, for the protection of infants and young children from severe pneumoniae and meningitis caused by H. influenzae b infection. [10]As a type 2 conjugation method, both the synthetic oligosaccharides and carrier proteins need to be modified to facilitate the conjugation.Though the native Cys residues (in disulfide form that need to be reduced to free thiols before conjugation) in the carrier proteins can serve as the conjugation partner, their low abundance limits the hapten loading.Thus, in general, more abundant Lys residues are labelled with thiol or maleimide, followed by the conjugation with correspondingly modified haptens.On the contrary to type 1 conjugation methods, the thiol-maleimide conjugation involves the possi-bility that the functional groups installed to the carrier proteins may not be fully conjugated with the modified haptens giving conjugates containing undesired "scars".Fortunately, though imperfect on molecular level, the "scars" do not affect the immunogenicity of the vaccine due to their short length and less exposure compared to the haptens, and the formation of "scars" can be minimized via optimization of the conjugation protocols.
10c] The crude sPRP-maleimide, after ultrafiltration through a membrane with cutoff 1 kD, was subjected to the conjugation with thiolated TT protein (32 thiol groups based on Ellman test), which was obtained via reaction with dithiobis(succinimidyl propionate (Lomant's reagent, DSP) followed by DTT reduction and ultrafiltration.The conjugation was conducted in PBS buffer (pH 7.2) at 4 to 8 °C using 44 equiv. of sPRP-maleimide to give conjugate 25 with sugar/protein ratio 1/2.6 w/w (~18 glycans per protein).In 2013, Wu et al. reported the synthesis of vaccine 26 against N. meningitidis W135. [75]In this case, the complex octasaccharide was modified with thiol group (40 equiv.) was   [76] The octasaccharide comprising two minimal repeating units was obtained via controlled enzymatic degradation of Ab-54149 exopolysaccharide (EPS) using phage derived ΦAB6 tailspike protein (ΦAB6TSP).The obtained octasaccharide has the same homogeneity as synthetic sample, which demonstrates the potential of this approach to provide haptens that are difficult to synthesize.The free reducing end was modified to amine by (NH 4 ) 2 CO 3 treatment (Kochetkov amination), followed by thiol installation.The conjugation with maleimide modified CRM197 (21 maleimide groups installed) using 40 equiv. of hapten-thiol species to give 27 with hapten loading 4.8, while the rest 16 maleimide groups were blocked by cysteine.In 2022, a similar approach was used to the synthesis of vaccine 28 against K. pneumoniae K2. [77] The octasaccharide/tetrasaccharide mixture obtained via enzymatic degradation of CPS was modified with thiol and conjugated to maleimide modified CRM197 to give 28 with hapten loading 7.4.The vaccine based on K1 CPS derived hexasaccharide was also described in this report.More examples using thiol-maleimide conjugation were summarized in Table 1 (part D). [78][79][80][81][82][83][84][85] Thioether Conjugation (Type 2) As another way to generate CÀ S bond linkage, the thioether conjugation via the S N 2 substitution of alkyl halide with thiol group has also been used in the glycoconjugate vaccine synthesis.Compared with the maleimide, alkyl bromide (normally α-bromoacetamide group on Lys residues) is less reactive towards nucleophiles and needs a higher pH (e. g., 8.0-9.0) to react with thiol. [86]Some examples are shown in Figure 8.In 2001, Jansen et al. reported the synthesis of vaccine 29 against S. pneumoniae based on the serotype 6B epitope. [87]The thiol modified tetrasaccharide (4000 equiv.) was conjugated to bromoacetylated KLH protein (prepared from KLH and Nsuccinimidyl bromoacetate) [88] following the protocol they developed before [89] to give conjugate with hapten loading 390.In 2010, Nifantiev and Pier et al. reported the preparation of glycoconjugate vaccine 30 based on the synthetic oligoglucosamine. [90]The β-(1!6)-poly-N-acetyl-D-glucosamine (PNAG) and deacetylated form (dPNAG) are surface glycans provided by several Gram-positive and Gram-negative pathogenic bacteria, including S. aureus, E. coli, Y. pestis, B. pertussis, and A. baumannii.Using short synthetic dPNAG mimetic (at least five glucosamine units) as hapten can generate vaccine with broad protection spectrum.The vaccine candidate 30 prepared by conjugating thiol modified pentasaccharide (57 equiv.) to bromoacetylated TT protein via thioether conjugation elicited strong immune response and gave much better opsonic killing effect of S. aureus than the one comprising N-acetylated hapten.In 2018, vaccine 31 against S. pneumoniae serotype 1 was developed by Seeberger et al. [91] The CPS derived zwitterion trisaccharide (68 equiv.) was conjugated to bromoacetylated CRM197 (bearing 18.4 bromide on average) to give conjugate with hapten loading 8.1.The thioether conjugation was also involved in the preparation of self-adjuvanting anti-C.albicans vaccine by Bundle et al., where the β-1,2-linked mannotriose hapten tethering to a 14-mer peptide Fba (derived from the N-terminal region of C. albicans fructose-bisphosphate aldolase) was conjugated to bromoacetylated TT protein. [92]

Click Conjugation (Type 2)
Since its discovery by Sharpless [93] and Meldal [94] , click chemistry (especially the Huisgen azide-alkyne 1,3-cycloaddition [95] ) has been developed into a robust tool for constructing molecular architectures and has been applied to almost all subdisciplines of chemistry, as well as biology and material science.Since both alkyne and azide are nonnative to proteins, the modifications of carrier proteins are needed to install azide or alkyne functionalities.The robustness of the azide-alkyne cycloaddition makes click chemistry a potential way to struct conjugate vaccines with improved homogeneity if the site-selective or site-specific azide/alkyne incorporation can be achieved.Currently, the application of azide-alkyne cycloaddition in synthetic glycoconjugate vaccine is not as popular as the conjugation methods discussed above, possibly due to the potential immunogenicity of the generated triazole structure in the linker and the safety issues from the contamination by Cu(I) catalysts. [96]Some examples are shown in Figure 9.

Other Conjugation Methods
Other type 1 conjugation methods include isothiocyanate conjugation, acyl azide conjugation, and o-phthalaldehyde (OPA) conjugation (Figure 10a).In 2016, Davis et al. reported the vaccine based on the core tetrasaccharide Hep 2 Kdo 2 , which is the common structural motif of LPS shared by several Gramnegative pathogenic bacteria like N. meningitidis, P. aeruginosa, and E. coli. [101]The synthetic tetrasaccharide with amine group installed at the reducing end was treated with CSCl 2 to give isothiocyanate, and the subsequent conjugation with DT  double mutant protein (200 equiv.isothiocyanate) at pH 9.0 gave conjugate 34 with hapten loading 4. In 2023, Field et al. reported the conjugation of chemoenzymatically synthesized β-1,2-oligomannosides to BSA. [102]The methyl ester group installed at the reducing end was treated with hydrazine monohydrate at 55 °C to give hydrazide, followed by activation by tert-butyl nitrite to form acyl azide.The conjugation of acyl azide (> 50 equiv.)to Lys residues at pH 9.1 gave conjugate vaccine candidate 35 with hapten loading 5.7.In 2021, Chen and Li et al. developed the first synthetic pseudaminic acid (Pse) based conjugate vaccine 36 against A. baumannii. [103]The 5NAc/ 7NAc Pse forming α-glycosidic linkage with PEG 4 spacer was modified by OPA via amide coupling, followed by the conjugation to CRM197 via the OPA-amine two-component reaction [104] under neutral conditions.The hapten loading 14.3 can be achieved by using 50 equiv.of OPA species.Conjugate 36 elicited immune response against Pse as well as the Pse containing glycan, which demonstrates the possibility of developing a single vaccine to cover several serotypes and even bacteria with shared key structure unit.
In addition to the type 2 conjugation methods described above, oxime and hydrazone conjugations [105] that have been well developed for bioconjugation were also applied in glycoconjugate vaccine studies (Figure 10b).In 2006, Pozsgay et al. reported the preparation of vaccine 37 against S. aureus based on the synthetic D-ribitol-1-phosphate oligomer (teichoic acid) hapten. [106]The oligomer was modified with a ketone functionality and subjected to the oxime conjugation with hydroxylamine modified BSA protein (~30 hydroxylamine per protein) at pH 7.4, giving conjugate with hapten loading 15.In 2013, Snapper et al. reported the preparation of vaccine 38 via conjugation of synthetic (oligo)glycerolphosphate hapten with TT protein. [107]The hydrazone conjugation was conducted at pH 6.0 in the presence of 10 mM aniline as catalyst.The hapten loading information was provided.

Summary and Outlook
The rapid progress in the synthetic glycoconjugate vaccine development in the past two to three decades and encouraging success of Quimi-Hib vaccine using synthetic oligosaccharide hapten demonstrate the great potential of this approach in combating infectious diseases caused by antibiotic resistant "bad-bugs".Compared to vaccines based on polysaccharide haptens isolated from pathogens, the structural homogeneity of the synthetic haptens and well-defined single site (reducing end in most cases) modifications not only eliminate the safety concern caused by contamination, but also significantly simplify the conjugate vaccine structure by avoiding the formation of crosslinked carbohydrate-protein network when multiple sites modified polysaccharides are used.Moreover, the accessibility of the well-defined hapten structures makes structure-immunogenicity relationship study and hapten optimization possible.
As a key step in the glycoconjugate vaccine preparation, the conjugation of oligosaccharide haptens to carrier proteins affects the hapten loading and conjugation sites, which leads to different immunological performance.In this review, the currently used conjugation methods in synthetic glycoconjugate vaccine development are summarized.Different chemical natures of the conjugation methods determine the hapten loading, site selectivity, reaction conditions (pH, temperature, and rate), and purification protocols.Since the hapten structures can affect conjugation behavior without good predictability, test and comparison of different types of conjugation methods in the available toolbox for every special case is important for success.Further development of the conjugation toolbox will provide more choices for researchers and increase the success rate.Meanwhile, some basic questions about conjugate site selectivity should be answered.
As discussed in the introduction part, the site of conjugation can affect immunogenicity.Achieving site-selective conjugation of haptens will allow us to fully understand immune response mechanism of glycoconjugate vaccines. [108]Thus, from the synthetic chemistry aspect, making "real" homogeneous vaccines by site-selective/site-specific conjugation of synthetic haptens is the eventual goal to pursue.Based on the pioneering explorations in polysaccharide conjugate vaccine, [109] two approaches are feasible.
The first approach is changing the conjugation sites to less abundant residues by new conjugation methods.Currently used conjugation methods mainly rely on the Lys residues.The high abundance of Lys residues in carrier proteins (e. g., 39 Lys in CRM197) and availability of Lys-selective labelling methods ensure smooth conjugation and enough hapten loading.However, achieving selectivity between Lys residues located at different positions is difficult and less studied. [98]Changing conjugation sites to less abundant Tyr residues (18 Tyr in CRM197) by Tyr-selective conjugation methods can improve the site-selectivity and product homogeneity. [97,110]Conjugation methods toward other residues like Cys, Trp, Arg and Met are worth testing in vaccine development. [111]In a recent endeavor, Adamo et al. site-specifically introduced a single ketone moiety by selectively reducing the C186-C201 disulfide bond and rebridging it with 1,3-dichloroacetone.The obtained CRM197-DCA was subjected to azide installation via oxime ligation and used in the conjugation with strained alkyne modified polysaccharide haptens. [112]he second approach is using bioengineered carrier proteins.In 2011, Davis et al. reported the generation of homogeneous glycoconjugate vaccine candidates via thioether linkage. [113]The alkene group was site-specifically introduced into subtilisin S156C mutant via Tamura's reagent mediated dehydroalanine (Dha) formation, or into viral capsid Qβ protein by genetic incorporating homoallylglycine residue.The synthetic pentasaccharide derived from K. pneumoniae armed with thiol on the reducing end was conjugated to the engineered two carrier proteins via Michael addition and thiol-ene reaction respectively.In 2019, Grandjean et al. reported the synthesis of homogeneous anti-S.pneumoniae glycoconjugate vaccine. [114]he four well exposed Lys residues (K237, K242, K247, K309) in carrier protein mPsaA (Pneumococcal surface adhesin A 21-309) were selected for single site mutation to generate the corresponding Cys-containing mutants.The four homogeneous vaccines formed by conjugation of tetrasaccharide hapten via thiol-maleimide reaction were tested in the immunization of mice for structure-immunogenicity study.In 2021, Wassil et al. reported the preparation of a novel 24-valent pneumococcal conjugate vaccine (VAX-24). [115]The bioengineered version of CRM197 (called enhanced CRM or eCRM) that the unnatural amino acid p-azidomethyl-L-phenylalanine (pAMF) was site specifically incorporated was used as carrier protein to allow site specific click conjugation of polysaccharide haptens.Other carrier proteins can be engineered in a similar manner to facilitate site specific conjugation.
We have witnessed the encouraging success of synthetic glycoconjugate vaccines in the past two to three decades.As a complicated project, successful vaccine development relies on the collaborative work from academia and pharmaceutical industry with different research backgrounds like chemistry, microbiology, and immunology.As synthetic chemists, we believe that further progress in the conjugation methods with the focus on both efficiency and selectivity will improve the immunological performance of synthetic glycoconjugate vaccines and help people to maintain advantage in the race with infectious diseases in the era of antibiotic resistance.

Zirong
Huang received her B.Sc. degree in chemistry from Guangxi Normal University, China, in 2017 and the M.Sc.degree in analytical chemistry from Xiamen University in 2020.She joined Prof. Sheng Chen's group as a Ph.D. candidate in 2021.Her current research is focused on the development of antibacterial synthetic glycoconjugate vaccines.Sheng Chen is a professor in the Department of Food Science and Nutrition at the Hong Kong Polytechnic University (polyU).Prof. Chen received both his B.Sc. degree in 1997 and M.Sc.degree in 2000 from the China Agriculture University.He received his Ph.D. degree in food microbiology at the University of Maryland in 2004.After postdoctoral training in the Department of Microbiology and Molecular Genetics at the Medical College of Wisconsin, he started independent academic career in 2009.His research lies on the interface of clinical study and basic science using multi-disciplinary approaches including genomics, genetics, biochemistry, cell biology and chemical biology with the goal of devel-oping novel therapies to combat bacterial antimicrobial resistance.Xuechen Li is a professor in the Department of Chemistry at the University of Hong Kong (HKU).Prof. Li received his B.Sc. degree in 1997 from Nankai University and M.Sc.degree in 1999 from University of Alberta in 2003.He received his Ph.D. degree in Harvard University in 2007.After doing postdoctoral research in Memorial Sloan Kettering Cancer Center, he joined HKU in 2009.His research interests include chemical protein synthesis, bioconjugate chemistry, carbohydrate chemistry, chemical biology and drug discovery.Han Liu received his B.Sc. (chemistry) and Ph.D. (organic chemistry) degrees from Peking University in 2005 and 2010, respectively.He did postdoctoral research at the University of Hong Kong (2010-2018) on carbohydrate/ peptide chemistry and Aarhus University (2018-2020) on natural product synthesis.He is currently a research assistant professor at the Department of Chemistry, the University of Hong Kong.His current research interests include total synthesis of peptide antibiotics and development of synthetic glycoconjugate vaccines.

Figure 1 .
Figure 1.Two mechanistic models of T cell activation by glycoconjugate vaccines.

Figure 2 .
Figure 2. General structure of glycoconjugate vaccines based on synthetic oligosaccharides and classification of conjugation methods.

Figure 10 .
Figure 10.Vaccines based on other conjugation methods.

Table 1 .
More examples of glycoconjugate vaccines using different conjugation methods.

Table 1 . continued
to the maleimide modified CRM197 (20 maleimide groups per protein) in PBS buffer at pH 7.4 to give conjugate 26 with hapten loading 2.8.In 2018, Wu et al. reported the synthesis of vaccine 27 against A. baumannii 54149.