Double conjugation strategy to incorporate lipid adjuvants into multiantigenic vaccines

Conjugation of multiple peptides by their N-termini is a promising technique to produce branched multiantigenic vaccines.


Introduction
The ability to develop safe vaccines using minimal microbial components has triggered rapid growth in research into peptide-based vaccines. 1However, the inability of peptides in isolation to stimulate the immune system is one of the key challenges in the development of peptide-based vaccines.Therefore, an adjuvant (immunostimulant) is necessary to stimulate a potent immune response against peptide epitopes. 2However, the use of adjuvants is usually associated with side effects and substantial toxicity that has limited the number of adjuvants approved for human use. 3 Only alum has been approved as a general human adjuvant, while just a few others were approved for particular vaccine formulation e.g.MF59, ASO3 and ASO4. 4 Unfortunately for anticancer vaccines, adjuvants that stimulate safe and effective cytotoxic T lymphocyte (CTL) responses are scarce. 5To overcome this problem, peptide vaccine research has turned its focus to the development of self-adjuvanting delivery systems.These vaccines combine peptide epitopes and immunostimulatory moieties (for example lipidic or polymeric entities) in a single covalently-linked conjugate, thereby ensuring co-delivery of the antigen to antigen presenting cells (APCs) activated by immunostimulatory moieties.This combined presentation helps to enhance vaccine potency and to avoid undesirable side effects that result from using classical adjuvants. 6very year, approximately 500 000 women are newly diagnosed with cervical cancer around the world, making it the second most common cancer among women.According to experimental and epidemiological studies, human papilloma virus (HPV) is the main cause of cervical cancer. 7Two high-risk genotypes, HPV types 16 (HPV-16) and 18 (HPV-18) are responsible for 70% of all cervical cancers. 8rophylactic vaccines against HPV infection help to reduce the incidence of cervical cancers through the generation of neutralizing antibodies and are only effective if administered before infection with HPV. 9 Hence, there is a strong demand for the development of effective therapeutic vaccines that are able to treat HPV-related cancers. 10The HPV genome encodes two types of proteins: early proteins (E1, E2, E4, E5, E6 and E7) and late proteins (L1 and L2).Expression of the E6 and E7 oncoproteins results in deregulation of the cell cycle, inactivating tumour suppressor gene products p53 and retinoblastoma protein (pRb) and leading to cancer. 10eptide-based strategies to develop therapeutic vaccines against HPV-associated cancers have shown promising outcomes in several early stage clinical trials. 10The choice of an appropriate peptide antigen is a crucial issue in the design of synthetic peptide vaccines.Therapeutic vaccines to treat cancer must elicit cellular immunity, thus must include CTL (CD8 + ) epitopes.1a A CTL epitope was identied in the HPV-16 E6 protein sequence (QLLRREVYDFAFRDL; E6 43-57 ) 10,11 and was previously shown to induce CTLs in vivo. 11,12Recently, our group showed that the 8Q min peptide, a small fragment of HPV-16 E7 protein (QAEPDRAHYNIVTF; E7 44-57 ), 9 that encodes CTL and Thelper cell epitopes, could reduce tumour growth and eradicate E7-expressing TC-1 tumour cells in mice through activation of CTLs 13 when administered with a self-adjuvanting delivery system.Therefore, these two CTL epitopes, E6 43-57 and 8Q min , were chosen as promising antigens for peptide vaccine development.We also recently demonstrated that anti-8Q min antibodies were not produced by mice vaccinated with the 8Q min epitope conjugated to the poly tert-butyl acrylate delivery system. 14t was reported that the orientation of antigens in a vaccine conjugate was very important for stimulating an immune response. 15Conjugation of different peptides via the C-terminus is valuable for the development of multiantigenic branched vaccines.Branched antigens tend to have increased stability to proteolysis, 16 and therefore a longer circulation time in the host, providing more opportunities to be taken up by APCs.As a result, these peptides can elicit stronger in vivo immune responses than linear peptides. 17e recently reported that modication of the 8Q min epitope from the E7 protein by replacing the C-terminal CCKCD sequence with SSKSD or SKKKK substantially diminished its immunogenicity.In contrast, deletion of the CCKCD sequence did not have any negative inuence on the epitope potency. 9These results suggest that the CTL epitope is only effective if conjugated to the vaccine delivery system via its Nterminus.
The attachment of a lipidic moiety to the N-terminus of an antigenic peptide to obtain amphiphilic vaccine molecules was previously reported.6c,6d,18 However, the N-terminal conjugation of two or more different unprotected epitopes to a vaccine delivery system have not yet been described (to the best of our knowledge).Thus we established a double conjugation synthetic technique to allow the conjugation of different unprotected peptides, E6 43-57 and 8Q min , via their N-termini in order to produce novel branched multiantigenic immunotherapeutics.

Results and discussion
We designed and synthesised immunostimulatory lipoalkynes 1-3 (Scheme 1a).These lipoalkynes were based on the structure of Pam2Cys (di-palmitoyl-S-glycerol cysteine), a well-characterised self-adjuvanting moiety that is widely used in experimental vaccine design. 19As Pam2Cys is a thio-1,2-diglyceride ester of palmitic acid, the new 1,3-diglyceride lipoalkynes 1-3 were designed by replacing the two ester linkages in Pam2Cys with two ether bonds to increase the stability of the compounds against esterases.The two long hydrocarbon chains in Pam2Cys were modied by substituting two methylene groups with oxygen atoms in two different positions as in lipid 1 and 2, to investigate the effect of increasing the polarity (and subsequently the aqueous solubility) on the adjuvanting effect of the resulting molecules. 20For control purposes, lipid 3 contained the same hydrocarbon chain as Pam2Cys was synthesised.In contrast to Pam2Cys, lipids 1-3 have no chiral center and therefore exist as single isomers.They carry an alkyne moiety, thereby allowing easy conjugation of an antigen through a copper-catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction.
Lipoalkynes 1-2 were synthesised using three straightforward steps (Scheme 1b), while lipoalkyne 3 required only two steps to be produced.Alcohols 6-7 were prepared from diols 4-5 in 42 and 44% yields, respectively, using alkyl bromide and a phase-transfer catalyst tetrabutylammonium iodide (TBAI) in presence of DMF as a solvent under sonication conditions.The sonication of a mixture of alcohols 6-8, powdered sodium hydroxide, epichlorohydrin, and tetrabutylammonium bromide (TBABr) provided the branched alcohols 9-11.Alcohols 9-11 were treated with propargyl bromide and sodium hydride to afford the lipoalkynes 1-3 in good yields.
A double conjugation strategy was developed to produce anticancer vaccine candidates.Two peptide epitopes were combined into a multiantigenic construct via thioether conjugation using an acryloyl peptide.The reaction was examined on two model short peptides (12 and 13), where one peptide carried both mercapto and azide groups at its N-terminus (12) and the other peptide had an acryloyl moiety attached to its N-terminus (13) (Scheme 2).
The mercapto-acryloyl conjugation conditions were optimised; $pH 7.3, 37 C, 14 h in the presence of denaturants (6 M guanidine) was found to be optimal.The product 14 of this conjugation was reacted with a model alkyne 15 producing the desired conjugate 16 (Scheme 2, Fig. 1 and 2).The ability of double conjugation strategy to be performed in a one pot reaction was also demonstrated (Scheme 3 and Fig. 3).
The new vaccine candidates, lipopeptides 24-26, were synthesised using the developed conjugation method.First, the Nterminal amine moieties of 8Q min and E6 43-57 were modied using stepwise SPPS.Fmoc-cysteine and azidoacetic acid were coupled to 8Q min to produce mercapto-azide derivative 21.The second peptide (E6 43-57 ) was modied with acrylic acid to afford acryloyl derivative 22.The two modied unprotected peptides (21 and 22) were then conjugated via a Michael addition mercapto-acrylate reaction to produce azide derivative 23.
A solution of mercapto-azide 21 (2 equiv.)and the acryloyl derivative 22 (1 equiv.)were gently shaken in denaturing buffer comprised of 6 M guanidine, 50 mM sodium phosphate, 20% acetonitrile, 5 mM EDTA, at $pH 7.3 to afford the azide derivative 23 in an excellent isolated yield of 90% (Scheme 4).The reaction was monitored by analytical HPLC and mass spectroscopy (Fig. 4).The second conjugation between the azide derivative 23 (1 equiv.)and the lipoalkynes 1-3 (1.5 equiv.) was achieved in degassed DMF under a nitrogen atmosphere using the CuAAC reaction in the presence of copper wire 6b for 4 hours at 50 C to produce the nal lipopeptides 24-26 in 49-87% isolated yields (Scheme 4).The nal products 24-26 were structurally well-dened, with only one stereoisomer present, and the synthesis was simple and reproducible.
Scheme 2 Model double conjugation through a Michael addition, between 12 and 13, followed by a CuAAC reaction, between 14 and 15.
Fig. 1 Optimising the conditions for mercapto-acryloyl conjugation between model mercapto-azide (12) that used in excess and acryloyl derivative (13) peptides (a) at 0 time; (b) at 3 h, using DMF as a solvent and two drops of DIPEA.New products started to form including the mercapto-acryloyl conjugation product (14) and the dimer of 12; (c) at 7 h in DMF/DIPEA the reaction was completed with the formation of peptide 14 and the dimer of 12 as major products with the remaining of the majority of the acryloyl peptide 13; (d) at 3 h, using guanidine buffer as a solvent ($pH 7.3) at 37 C, new products formed, the mercaptoacryloyl conjugation product (14) together with the dimer of 12; (e) at 14 h in a guanidine buffer, the reaction was completed by formation of peptide 14 as a major product together with the dimer of 12 and complete consumption of the acryloyl peptide 13.The reaction progress was monitored by HPLC and the products were detected by mass spectrometry.The therapeutic effect of the multiantigenic conjugates on established HPV tumour was evaluated in a mouse model, 6-8 week old, female C57BL/6 mice.The design of compounds 1-3 was based on the structure of Pam2Cys, hence Pam2Cys conjugated to the 8Q min /E6 43-57 epitopes was used as a control (29) (Scheme 6).The therapeutic importance of incorporating two epitopes in one molecular entity (24-26)  was explored by synthesising compounds 27 and 28, which were comprised of lipid 1 conjugated to 8Q min or E6 43-57 , respectively.At day zero, mice (8/group) were implanted in the side ank with TC-1 tumour cells expressing the E6/E7 oncoproteins. 21On day 3 mice were immunised with either lipopeptides 24-26 (100 mg/100 mL sterile PBS), a physical mixture of lipid 1 conjugated with 8Q min (27) and lipid 1 conjugated with E6 43-57 (28) (100 mg/100 mL sterile PBS, 1 : 1) (Scheme 5), 8Q min /E6 43-57 epitopes conjugated with Pam2Cys (29) (100 mg/100 mL sterile PBS) as a positive control, or PBS (100 mL) as a negative control.The Kaplan-Meier survival curve (Fig. 5a) showed that all of the mice treated with PBS were euthanised due to tumour burden by day 45.In contrast, mice treated with lipopeptide 24 and 25 demonstrated 38% (3 out of 8 mice) and 25% (2 out of 8 mice) survival rates, respectively, which was signicantly better than that for mice treated with the positive control (8Q min /E6 43-57 -Pam2Cys (29), 0% survival, 0 out of 8 mice).Among tested groups, the slowest tumour growth was observed in mice immunised with vaccine candidate 24 (Fig. 6a).The physical mixture of 27 and 28 did not slow down tumour growth signicantly and only one mouse treated with the mixture survived to the end of the experiment.We proposed that the physical mixture of 27 and 28 would allow each epitope to be taken up by different APCs, thus the immune stimulating effect of Thelper epitope present in 8Q min may not enhance the immune response against the E6 43-57 epitope.It was reported that the co-recognition of T-helper and CTL epitopes by the same APC was essential for the efficient stimulation of cellular immunity. 22Interestingly, the biological study  showed that Pam2Cys analogue (29) and the most hydrophobic vaccine candidate 26 induced very weak antitumour responses.In tumour challenge, 0/5 and 1/5 mice survived on day 60 for 29 and 26, respectively (Fig. 5a) despite the wellproven ability of Pam2Cys to induce cellular immune responses. 23This might be explained by the formation of large aggregates (>5 mm in diameter) by conjugates 26 and 29 while compounds 24 and 25 formed particles of submicron size (0.3-0.8 mm as measured by dynamic light scattering) under aqueous conditions.This observation is in the agreement with well-known phenomena that the immune responses are highly dependent on the vaccine particle size. 2 This size difference may have arisen because the presence of oxygen atoms in the hydrocarbon chain in both lipids 1 and 2 increased the solubility of the latter compounds (24 and 25).
Compound 29 (which bore a Pam2Cys moiety) induced unexpectedly weak antitumour responses, therefore two additional independent experiments (with 5 + 8 mice per group) were performed to further investigate the antitumour potency of the lead vaccine candidate.Incomplete Freund's adjuvant (Montanide ISA51) was chosen as an adjuvant in an emulsion with 8Q min and E6 43-57 epitopes for formulation of the positive control.

Conclusions
We established a synthetic double conjugation pathway to develop multiantigenic lipopeptide conjugates as self-adjuvanting therapeutic vaccine candidates to treat HPV-related cancers.The method involved a Michael addition mercaptoacryloyl reaction between two unprotected peptides followed by an azide-alkyne click reaction to give the nal lipopeptide products.Three novel lipidic self-adjuvanting moieties were synthesised, conjugated with the multiantigenic branched  peptide and were found to stimulate signicantly better survival in an in vivo murine HPV model than mice treated with the Pam2Cys analogue 29, without an external adjuvant and aer only a single immunization.Our double conjugation strategy provided an overall yield 50-80%; can be applied to a wide variety of synthetic applications; was simple to perform; and can be applied on unprotected peptides.This strategy could also be used to rapidly produce libraries of different vaccine constructs from a small selection of starting components (e.g. a variety of epitopes and adjuvants).It is anticipated that this technique will be widely used in the chemical synthesis of branched multiantigenic peptides and self-adjuvanting vaccines.
Synthesis of azidoacetic acid (N 3 CH 2 CO 2 H).Azidoacetic acid was synthesised using a similar method to the published procedure. 27Sodium azide (6.0 g, 92.3 mmol, 3.0 equiv.) was dissolved in H 2 O (10 mL) and bromoacetic acid (4.3 g, 30.8 mmol, 1.0 equiv.) was added.The reaction mixture was covered and protected from light with aluminum foil.The reaction was stirred continuously in an ice bath for 24 h and subsequently acidied with 32% HCl (10 mL).The product was then extracted with Et 2 O (4 Â 50 mL), dried over anhydrous MgSO 4 and the solvent was evaporated under vacuum.The nal product was obtained as a colorless oil (2.95 g, 95%) aer prolonged evaporation under vacuum to remove organic solvent and the last traces of water. 1 H NMR (300 MHz, CDCl 3 ) d 3.98 (s, 2H), 10.60 (br s, OH, 1H). 13C NMR (100 MHz, CDCl 3 ) d 173.7, 50.0.
Synthesis of multiantigenic peptide azide ( 23) through mercapto-acryloyl conjugation.A mixture of the two peptide epitopes acryloyl E6 43-57 (22) (7.2 mg, 3 mmol, 1.0 equiv.)and 8Q min mercapto-azide (21) (13.4 mg, 6 mmol, 2 equiv.)was dissolved in a guanidine buffer at $pH 7.3.The reaction mixture was incubated at 37 C for 48 h.The progress of the reaction was monitored by analytical HPLC until the acryloyl E6 43-57 (22) was completely consumed.The reaction mixture was puried using a semi-preparative HPLC on a C-18 column (20-60% solvent B over 60 min).Aer lyophilization, the pure azide derivative 23 was obtained as an amorphous white powder.The product was detected using analytical HPLC analysis (C-4 column, Method A), t R ¼ 21.8 min, purity > 97% and (C18 column, Method A), t R Synthesis of vaccine candidate lipopeptide 24.A mixture of azide derivative 23 (3.3 mg, 7.5 Â 10 À4 mmol, 1 equiv.)and the lipoalkyne 1 (0.6 mg, 11.3 Â 10 À4 mmol, 1.5 equiv.) was dissolved in DMF (1 mL), and copper wire (80 mg) was added.The air in the reaction mixture was removed by nitrogen bubbling.The reaction mixture was covered and protected from light with aluminum foil and stirred at 50 C under nitrogen.The progress of the reaction was monitored by analytical HPLC (C-4 column) and ESI-MS until the peptide 23 was completely consumed aer 4 h.The reaction mixture was puried using a semi-preparative HPLC on a C-4 column (35-75% solvent B over 60 min).Aer lyophilization, the pure lipopeptide 24 was obtained as an amorphous white powder.Compound 24 was analysed by HPLC (C-4 column, Method A) t R ¼ 29.9 min, purity > 97% (detected by UV at 214 nm) and t R ¼ 30.0 min, purity > 96% (detected by evaporative light scattering detector).Yield: (3.2 mg, 87%).
Synthesis of vaccine candidate lipopeptide 25.A mixture of azide derivative 23 (3.0 mg, 0.7 mmol, 1 equiv.)and the lipoalkyne 2 (3.0 mg, 1.8 mmol, 2.5 equiv.) was dissolved in DMF (1 mL), and copper wire (60 mg) was added.The air in the reaction mixture was removed by nitrogen bubbling.The reaction mixture was covered and protected from light with aluminum foil and stirred at 50 C under nitrogen.The progress of the reaction was monitored by analytical HPLC (C-4 column) and ESI-MS until the peptide 23 was completely consumed aer 3 h.The reaction mixture was puried using a semi-preparative HPLC on a C-4 column (40-80% solvent B over 60 min).Aer lyophilization, the pure lipopeptide 25 was obtained as an amorphous white powder.Compound 25 was analysed by HPLC (C-4 column, Method A) t R ¼ 29.9 min and t R ¼ 36.2 min (C-4 column, Method B), purity > 97% (detected by UV at 214 nm).Yield: (2.7 mg, 80%).
Synthesis of N-uorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)cysteine (Fmoc-Dhc-OH). 28As shown in Scheme 6, a solution of uorenylmethoxycarbonyl-N-hydroxysuccinimide (1.15 g, 3.5 mmol) in acetonitrile (10 mL) was added to a solution of Dhc-OH (0.8 g, 3.5 mmol) in 9% sodium carbonate (10 mL).The reaction mixture was stirred at room temperature.Aer 2 h, water (100 mL) was added and the solution was acidied to pH 2 with concentrated hydrochloric acid and then extracted with ethyl acetate (3 Â 100 mL).The combined organic layers were washed with water (2 Â 50 mL) and brine (2 Â 50 mL), dried over anhydrous MgSO 4 and evaporated in vacuo to give sticky colourless solid.The crude product was puried with a preparative RP-HPLC on C-18 column with a 25-45% Synthesis of Pam2Cys-alkyne.Pam2Cys-alkyne was synthesised following the general manual stepwise SPPS HATU/ DIPEA Fmoc-chemistry procedure (Scheme 6).The attachment of Fmoc-Dhc-OH was achieved by dissolving a mixture of Fmoc-Dhc-OH (3 equiv.),DIC (3 equiv.)and HOBt (3 equiv.) in DMF (2 mL) at 0 C for 5 min.The activated species was then added to the resin and le to couple for 4 h, followed by a thorough wash with DMF and DCM.The S-glycerol-cysteine hydroxyl groups were then palmitoylated by addition of palmitic acid (20 eq.) activated with DIC (25 eq.) and 4-(dimethylamino)pyridine (DMAP; 2 eq.) in DCM.The reaction was le to complete overnight, and the resin subsequently washed with DCM.The 3-Mtt protecting group on the C-terminal lysine was then removed by using a mixture of 1% TFA and 5% TIPS in DCM (25 Â 5 min) followed by washing with DCM and DMF.The Dhc-associated Fmoc group was then removed by using 2.5% (w/v) DBU in DMF (3 Â 5 min), followed by washing with DMF, DCM, and drying under vacuum.The crude product was puried by a preparative RP-HPLC on C-4 column with a 40-80% solvent B gradient over 60 min.HPLC analysis (C-4 column, Method A): t R ¼ 33.5 min, purity > 95%.Yield: 20%.ESI-MS: m/z 1351.2 (calc 1351.9)[M + H] + ; 676.4 (calc 676.5) [M + 2H] 2+ ; MW 1350.9.

Particle size measurement
The nal constructs of lipopeptides 24-26 and 29 were dissolved in sterile PBS using a vortex until a homogenous solution was obtained.The particle sizes were measured by dynamic light scattering.The measurements were repeated at least ve times.All compounds formed particles with rather high polydispersity.Compound 24 and 25 formed submicron particles (24: 450-750 nm, PDI ¼ 0.25-0.55 and 25: 350-550 nm, PDI is 0.20-0.40).Compounds 26 and 29 formed large aggregates (visible to the naked eye) with sizes above the upper detection level of the instrument (>5 mm).
Tumor challenge experiments.To test the efficacy of lipopeptide 24 conjugate as a therapeutic vaccine against established tumours, groups of C57BL/6 female mice (8/group) were rst challenged subcutaneously in the right ank with 1 Â 10 5 per mouse of TC-1 tumour cells.On the third day aer tumour challenge, the mice were injected subcutaneously at the tail base with: 100 mg of lipopeptide 24-26 conjugates in a total volume of 100 mL of sterile-ltered PBS; a mixture of lipid 1 conjugated with 8Q min (27) and lipid 1 conjugated with E6 43-57 (28) (100 mg/100 mL sterile PBS, 1 : 1); 8Q min /E6 43-57 -Pam2Cys (29) (100 mg/100 mL sterile PBS) as a positive control; or 100 mL PBS as a negative control.Each mouse received a single immunization only.The size of the tumour was measured by palpation and calipers every two days and reported as the average tumour size across the group of ve mice or as tumour size in individual mice. 30Tumour volume was calculated using the formula V (cm 3 ) ¼ 3.14 Â [largest diameter Â (perpendicular diameter) 2 ]/6.30b The mice were euthanised when tumour reached 1 cm 3 or started bleeding to avoid unnecessary suffering.
The second tumor challenge study was executed with 24 and antigens administered with commercial adjuvant, in the similar manner as described above.Two independent experiments were performed with total 13 C57BL/6 mice per group (5 + 8 mice per group) and the list of the immunization compounds was: lipopeptide 24; 30 mg of a mixture of 8Q min and E6 43-57 emulsied in a total volume of 100 mL of Montanide ISA51 (Seppic, France)/ PBS (1 : 1, v/v) as a positive control; and PBS as a negative control.

IFN-gamma ELISPOT assays
Splenocytes were harvested from the spleens of naïve and immunised mice and depleted of red-blood cells using Red Blood Cell Lysing Buffer (0.155 M ammonium chloride in 0.01 M Tris-HCl buffer, Sigma).Splenocytes were then resuspended in RPMI (Sigma) supplemented with 20% FCS (Sigma), 100 U mL À1 penicillin and 100 mg mL À1 streptomycin, and 50 mM bmercaptoethanol.The cells were plated at 5 Â 10 5 cells per well in triplicate in ELISPOT plates (Millipore Biotec) which had been previously coated with 5 mg mL À1 IFN-g capture mAb (clone 14-7313-85 eBioscience) in PBS at 4 C overnight and then blocked with RPMI/20% FCS at room temperature for 3 hours.E7 (RAHYNIVTF) and E6 (YDFAFRDL) peptides (10 mg mL À1 ) were added alongside 10 ng mL À1 rhIL-2 (R&D systems) to a nal volume of 200 mL per well.Plates were incubated for 18 hours at 37 C, washed, and a biotinylated IFN-g detection Ab (2 mg mL À1 ; clone R4-6A2; eBioscience) in 1% BSA added at room temperature for 3 hours.Aer washing, a streptavidin-HRP complex (DakoCytomation) in 1% BSA was added for 1 h at room temperature, plates were washed again, and bound cytokine was visualised with 3-amino-9-ethylcarbozole (Calbiochem).Spots were counted with an ELISPOT reader (Autoimmun Diagnostika).An "irrelevant" lipopeptide (KQAEDKVKASREAKKQVEKALEQLEDKVKconjugated with lipid 1) was used as a control in this experiment.Group A streptococcal B-cell epitope (J14) was selected as the irrelevant peptide. 31tatistical analysis.All data were analysed using GraphPad Prism 5 soware.Kaplan-Meier survival curves for tumour treatment experiments were applied.Differences in survival treatments were determined using the log-rank (Mantel-Cox) test, with p < 0.05 considered statistically signicant.

Fig. 2 A
Fig. 2 A copper-catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) model reaction between model conjugation product (14) and model alkyne (15) in DMF in presence of Cu wire at 50 C under a nitrogen atmosphere (a) at 0 time; (b) at 1 h the reaction was completed by the complete consumption of 14 and formation of the model CuAAC product 16.

Fig. 3
Fig. 3 One pot double conjugation model reaction.Mercaptoacryloyl conjugation between mercapto-azide (17) and acryloyl derivative (18) at $pH 7.3 (guanidine buffer), 37 C (a) at 0 time; (b) at 72 h the reaction was completed by the complete consumption of 17 and formation of the conjugation product (19) together with the disulfide dimer of 17.In a one pot reaction, a CuAAC model reaction between model conjugation product (19) and model alkyne (15) in guanidine buffer in presence of Cu wire at 50 C under a nitrogen atmosphere (c) at 0 time; (d) at 1 h the reaction was completed by the complete consumption of 19 and formation of the model CuAAC product 20.The reaction progress was monitored by HPLC and the products were detected by Mass spec.

Fig. 4
Fig. 4 Mercapto-acryloyl conjugation between mercapto-azide (21) and acryloyl derivative (22) at pH 7.5, 37 C (a) at 0 time; (b) at 48 h the reaction was completed by the complete consumption of 22 and formation of the multiantigenic conjugation product (23) together with the dimer of 21.The reaction progress was monitored by HPLC and the products were detected by MS.