The Use of Electrochemical Voltammetric Techniques and High-Pressure Liquid Chromatography to Evaluate Conjugation Efficiency of Multiple Sclerosis Peptide-Carrier Conjugates

Recent studies have shown the ability of electrochemical methods to sense and determine, even at very low concentrations, the presence and quantity of molecules or analytes including pharmaceutical samples. Furthermore, analytical methods, such as high-pressure liquid chromatography (HPLC), can also detect the presence and quantity of peptides at very low concentrations, in a simple, fast, and efficient way, which allows the monitoring of conjugation reactions and its completion. Graphite/SiO2 film electrodes and HPLC methods were previously shown by our group to be efficient to detect drug molecules, such as losartan. We now use these methods to detect the conjugation efficiency of a peptide from the immunogenic region of myelin oligodendrocyte to a carrier, mannan. The HPLC method furthermore confirms the stability of the peptide with time in a simple one pot procedure. Our study provides a general method to monitor, sense and detect the presence of peptides by effectively confirming the conjugation efficiency. Such methods can be used when designing conjugates as potential immunotherapeutics in the treatment of diseases, including multiple sclerosis.


Introduction
Voltammetric techniques, including differential pulse voltammetry (DPV) and cyclic voltammetry (CV), as well as high-performance liquid chromatography (HPLC), were applied to identify and detect a peptide to its conjugated carrier. This study describes for the first time an alternative, fast, low cost and reliable method for the adequate and reliable determination of an active pharmaceutical ingredient (API) in the biocompatible matrix. The performance of the voltammetric techniques is strongly dependent on the performance of the working electrode used. Film electrodes, such as the graphite/SiO 2 used in this study, are being used in electrochemistry, as it has a number of advantages over the standard metallic and glass carbon electrodes. These include ease of manufacture requiring lower temperatures, low cost, the high surface area that could be rapidly renovated, simple handling, Sodium metasilicate (Na 2 SiO 3 ) (SiO 2 , 50-53%), NaH 2 PO 4 , mannan isolated from yeast cells (Saccharomyces cerevisiae), potassium ferricyanide, ferrocyanide, and potassium chloride were obtained from Sigma Aldrich Chemie GmbH (Taufkirchen, Germany). MOG  and MOG [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] peptides were supplied by NewDrug S.A., Patras Science Park, Greece and purchased from China peptides Inc. The peptide analogue (Lys-Gly) 5 -MOG  , referred as (KG) 5 -MOG  , was synthesized using standard peptide chemistry techniques and previously published by our group. Briefly, Fmoc/tBu methodology was used which included 2-chlorotrityl chloride resin (CLTR-Cl) and N a -Fmoc (9-fluorenylmethyloxycarboxyl) side chain protected amino acids [43,44]. The purity of the peptides were shown to be >97% by analytical HPLC. Graphite powder (synthetic, APS 7-11 µm, 99%) was obtained from Alfa Aesar. Soda lime glass slides (75 mm × 25 mm × 1.1 mm), with 15 Ohm/sqr Indium Tin Oxide (ITO) coating were obtained from PsiOTec, UK. All chemicals were of analytical grade and used without the need for further purification. All solutions were prepared in deionized water with resistance R = 18 MΩ cm.

Graphite/SiO 2 Film Electrodes Preparation
The graphite/SiO 2 film electrodes were prepared as described [2,3]. Briefly, silicate liquid polymer (50% Na 2 SiO 3 ; pH 12-13) was gently mixed with 20% graphite powder at 23 • C, until the mixture became homogeneous and acquired a "sticky" texture. The mixture underwent ultrasonication for 2 min for the graphite powder to be fully soluble, and 100 µL of the silicate/graphite suspension were applied on the surface of a conductive ITO glass slide using the "Doctor Blade" technique. Prior to the deposition of the silicate/graphite suspension, the ITO glass slides were cleaned in a detergent solution using an ultrasonic bath for 15 min, and then rinsed with 18 MΩ distilled water and ethanol. Each glass slide was masked with 3M Magic Scotch tape (thickness 62.5 µm; type 810), in order to control the width and the thickness of the mixture spread area. For each graphite/SiO 2 film deposition, one layer of tape was used which provided a size 1 × 1 cm 2 and film thickness of~66 µm. The films were allowed to dry for 30 min in a class 4000 room, prior to placing them in a preheated oven (330 • C) for 100 min. If required, the liquid suspension could be stored in an insulated flask at 25 • C for later usage. The resulting ITO substrates with the deposited graphite/SiO 2 films were cut in 10 mm × 25 mm pieces before use.

Characterization of Graphite/SiO 2 Film Electrodes
Field emission scanning electron microscopy (FE-SEM) using an FEI inspect microscope (25 kV) was used to determine morphology and thickness of the Graphite/SiO 2 film. The films were prepared by AU sputtering to increase the conductivity of the samples. Energy dispersive spectroscopy EDS was also used for the elemental analysis of the Graphite/SiO 2 /ITO films.

Preparation of (KG) 5 -MOG 35-55 Peptide
MOG  agonist peptide was synthesized in our labs, >97% purity, with (KG) 5 extended at the N-terminus of the peptide. Peptide was prepared using our methods, either by coupling, catalyzed by microwave radiation in a CEM Liberty microwave system or by using the conventional step by step procedure by solid phase peptide methods (as described in [45]). (KG) 5 -MOG  peptide was also purchased by China Peptides Inc. In house synthesized peptides and purchased peptides were confirmed by HPLC and Mass Spectroscopy for purity and identity.

Preparation of Oxidized Mannan
Mannan (14 mg) was dissolved in 1 mL phosphate buffer (0.1 M sodium phosphate, pH 6.0), and was oxidized using 0.1 M sodium periodate and incubated at 4 • C for 1 h, after which 10 µL ethanediol was added for 30 min at 4 • C. Oxidized mannan (OM) was passed through a PD-10 column (Sigma Aldrich Chemie) pre-equilibrated in sodium bicarbonate buffer (sodium carbonate: Sodium bicarbonate, pH 9.0). Two ml of OM fraction (7 mg/mL) was collected and kept in the dark.

Conjugation of Oxidized Mannan to Peptide
To the OM fraction (2 mL; 7 mg/mL, sodium bicarbonate pH 9.0 buffer), 1 mg of (KG) 5 -MOG  peptide was added and allowed to react overnight in the dark at 23 • C. A list of peptides and conjugates are summarized in Table 1.

Monitoring of Conjugation by HPLC
We used a Waters 2695 HPLC (Alliance) system with a photodiode array detector equipped with a Lichrosorb RP-18 reversed phase analytical column (C18 35 µm, 4.6 × 50 mm PIN 186003034). Analysis was achieved with stepped linear gradient of solvent A (0.08% TFA in H 2 O) and in solvent B (0.08% TFA in 100% acetonitrile) for 30 min with a flow rate 3 mL/min. The conjugation of OM with (KG) 5 -MOG  peptide was evaluated by HPLC. The (KG) 5 -MOG 35-55 HPLC peak disappeared within six hours indicating completion of conjugation to OM.

Electrochemical/Electrocatalytic Measurements
Electrochemical measurements were conducted using an Autolab PGStat-101 potentiostat (Metrohm, Utrecht, The Netherlands). The electrochemical cell comprised of a 10 mL, three-electrode stirring glass cell with a Teflon cap, a platinum mesh flag as the counter electrode, a Ag/AgCl/KCl sat reference electrode and a Graphite/SiO 2 film on ITO conducting glass as the working electrode. The electrolyte contained a solution of NaH 2 PO 4 (10 mM; pH 7.0), which was deoxygenated with argon prior to any measurements and an argon atmosphere was kept throughout the measurements. The DPV measurements took place in a potential range between −1 to +0.05 V. The optimized parameters of DPV correspond to a step potential at 5 mV, amplitude of 50 mV, modulation time of 25 ms with scan rate 100 mV s −1 and a frequency of 50 Hz. All potentials are reported against Ag/AgCl and all experiments were carried out at 23 • C.

FE-SEM Characterization
The general thickness and surface morphology of the graphite/SiO 2 films were demonstrated by FE-SEM. The top-view of the FE-SEM image (Figure 1a) shows that the surface of the graphite/SiO 2 film is rough and non-uniform with many wrinkles. It exhibits increased porosity and a high effective surface area. Figure 1b presents the cross section of a graphite/SiO 2 film electrode, with an estimated film thickness of~65 µm as set by the adhesive tape used; the EDS for a graphite/SiO 2 film carried out during Brain Sci. 2020, 10, 577 5 of 13 the FE-SEM analysis is shown in the Supplementary Materials (SM, Figure S1). The characteristic peaks of Na, O, and Si, due to the use of silicate glue (Na 2 SiO 3 ), are presented in high intensity, thus, the peak of C is presented in lower intensity. Hence, the results validate the reduced concentration of carbon in the mixture used for the fabrication of the graphite/SiO 2 films.
Brain Sci. 2020, 10, x FOR PEER REVIEW 5 of 14 intensity, thus, the peak of C is presented in lower intensity. Hence, the results validate the reduced concentration of carbon in the mixture used for the fabrication of the graphite/SiO2 films.

UV Characterazation of (KG)5-MOG35-55 Peptide with Increasing Amounts of OM
It is known that most peptides exhibit strong absorbance at around 280 nm, due to aromatic amino acids (tyrosine and tryptophan) or disulfide bonds in the peptide sequences [46,47]. Figure 2 shows the UV-vis spectra of (KG)5-MOG35-55 with increasing amounts of OM. The increase of absorbance at 280 nm confirms the conjugation of MOG35-55 peptide to OM. The intensity of the absorption peak at 280 increases until all of the free peptide in solution is conjugated to the OM. It should be noted that the conjugate of (KG)5-MOG35-55 with OM took place in solution and not on the surface of the graphite/SiO2 film electrode as due to its non-transparency it is impossible to monitor the conjugation process on its surface. All the UV-visible absorption spectra of the peptide was recorded using a Shimadzu UV-1800 spectrophotometer. Absorbance (a.u.)

Electrochemical Analysis Showing Conjugation of (KG)5-MOG35-55 to OM
Electrochemical characteristics of the graphite/SiO2 film electrode were investigated by CV. Figure 3a shows the electrochemical behavior of a bare graphite/SiO2 film electrode in a solution of

UV Characterazation of (KG) 5 -MOG 35-55 Peptide with Increasing Amounts of OM
It is known that most peptides exhibit strong absorbance at around 280 nm, due to aromatic amino acids (tyrosine and tryptophan) or disulfide bonds in the peptide sequences [46,47]. Figure 2 shows the UV-vis spectra of (KG) 5 -MOG  with increasing amounts of OM. The increase of absorbance at 280 nm confirms the conjugation of MOG  peptide to OM. The intensity of the absorption peak at 280 increases until all of the free peptide in solution is conjugated to the OM. It should be noted that the conjugate of (KG) 5-MOG  with OM took place in solution and not on the surface of the graphite/SiO 2 film electrode as due to its non-transparency it is impossible to monitor the conjugation process on its surface. All the UV-visible absorption spectra of the peptide was recorded using a Shimadzu UV-1800 spectrophotometer.
Brain Sci. 2020, 10, x FOR PEER REVIEW 5 of 14 intensity, thus, the peak of C is presented in lower intensity. Hence, the results validate the reduced concentration of carbon in the mixture used for the fabrication of the graphite/SiO2 films.

UV Characterazation of (KG)5-MOG35-55 Peptide with Increasing Amounts of OM
It is known that most peptides exhibit strong absorbance at around 280 nm, due to aromatic amino acids (tyrosine and tryptophan) or disulfide bonds in the peptide sequences [46,47]. Figure 2 shows the UV-vis spectra of (KG)5-MOG35-55 with increasing amounts of OM. The increase of absorbance at 280 nm confirms the conjugation of MOG35-55 peptide to OM. The intensity of the absorption peak at 280 increases until all of the free peptide in solution is conjugated to the OM. It should be noted that the conjugate of (KG)5-MOG35-55 with OM took place in solution and not on the surface of the graphite/SiO2 film electrode as due to its non-transparency it is impossible to monitor the conjugation process on its surface. All the UV-visible absorption spectra of the peptide was recorded using a Shimadzu UV-1800 spectrophotometer. Absorbance (a.u.)

Electrochemical Analysis Showing Conjugation of (KG)5-MOG35-55 to OM
Electrochemical characteristics of the graphite/SiO2 film electrode were investigated by CV. Figure 3a shows the electrochemical behavior of a bare graphite/SiO2 film electrode in a solution of

Electrochemical Analysis Showing Conjugation of (KG) 5 -MOG 35-55 to OM
Electrochemical characteristics of the graphite/SiO 2 film electrode were investigated by CV. Figure 3a shows the electrochemical behavior of a bare graphite/SiO 2 film electrode in a solution of 0.1 M KCl and 5 mM of [Fe(CN) 6 ] 3−/4− through CV in the potential range of +1 to −1 V at different scan rates. Figure 3b shows the currents (anodic and cathodic) from the plots of I vs. square root of scan rate (v 1/2 ). Straight lines form for both the anodic and cathodic currents, confirming that a diffusional process has occurred in the reaction of ferrocyanide/ferricyanide. In addition, these results confirm that fast electron transfer occurs on the Graphite/SiO 2 film electrode due to its increased conductivity and surface area. In order to calculate the electroactive surface area of the film electrode, the Randles-Sevcik equation was used [36]: where i p corresponds to the maximum current (in Amperes), n is the number of electrons transferred and v is the scan rate (mV s −1 ) and thus the electroactive surface area of the graphite/SiO 2 was estimated to be 0.0039 cm 2 .
Brain Sci. 2020, 10, x FOR PEER REVIEW 7 of 14   The electrochemical behavior of the graphite/SiO 2 film electrode was then investigated in the presence and absence of the MS myelin epitope peptide vaccine (OM-(KG)5-MOG  ). Figure 3c shows the effect of scan rate of a bare graphite/SiO 2 electrode, before the detection of the OM-(KG)5-MOG  , at a scan rate range of 0.01 to 0.1 V s −1 . All electrochemical experiments were performed in a peptide free, anaerobic 10 mM NaH 2 PO 4 (pH 7.0). The bare graphite/SiO 2 film electrode shows the characteristic charging/de-charging currents, and no cathodic or anodic peaks are observed even at the slowest scan rate (0.01 V s −1 ). One of the advantages of using graphite paste electrodes is the increased conductivity, which allows a broader study of redox reactions occurring at very high or low biases (ranging from +1 V to −1 V). Further, the slower scan rate applied, the smaller the resulting current is obtained. Figure 3b, on the other hand, showing the CVs of OM-(KG) 5 -MOG 35-55 on the graphite/SiO 2 film electrode, exhibits not only the characteristic charging/discharging currents assigned to electron injection into sub-band gap/conduction band states of the graphite/SiO 2 electrode, but also two reduction peaks around −0.22 V and −0.67 V and a broad re-oxidation peak at −0.1 V.
The redox peak currents were shown to be proportional to the scan rate, characteristic of quasi-reversible behavior. The rate of reaction between the graphite/SiO 2 electrode and the conjugate, OM-(KG) 5 -MOG  was not fast enough to maintain equal concentrations of oxidized and reduced species at the surface of the electrode. In addition, the CV responses were shown to be stable, with the waveforms being unperturbed after being scanned several times, whilst no other consumption of the complex occurred nor other undesirable reactions in the phosphate buffer took place.
In Figure 3d, the two cathodic peaks at −0.27 V and −0.7 V and the wide anodic peak approximately at −0.1 V observed are due to the presence of the OM-(KG) 5 -MOG  . The two cathodic peaks correspond to the linker molecule (KG) 5 used to conjugate the MOG 35-55 peptide to OM, that contains 5 lysines and 5 glycines to its structure. Thus, the cathodic peaks attributed to the presence of lysines. On the other hand, the wide oxidation peak occurred probably due to superfluity of the free (KG) 5 -MOG 35-55 peptide that was not able to conjugate to OM and created the final complex of the OM-(KG) 5 -MOG  conjugate.
The CVs of the constituents of the OM-(KG) 5 -MOG  conjugate are shown in Figure 4. According to Figure 4a, as mentioned earlier, the bare graphite/SiO 2 film electrode exhibited no reduction or oxidation peaks which is consistent with the currents being limited by the graphite conductivity at the voltage biases reported herein. On the other hand, the CV of the film electrode in the presence of mannan in 0.1 M buffer exhibited an oxidation peak at approximately 0.5 V, and the CV of the film electrode in the presence of 0.002 mg/mL OM displayed a slight cathodic peak at −0.56 V and the characteristic anodic peak at −0.1 V. At the same time, the electrochemical behavior of peptides MOG  and MOG 37-55 were examined. The main difference between these two peptides is that the MOG 35-55 peptide contained and additional linker with 5 lysines (KG) 5 , whilst the MOG 37-55 peptide included a linker, which only contained 1 lysine. This was confirmed in Figure 4b, which displays the CVs of the Graphite/SiO 2 film electrode in the presence of each peptide. The two cathodic and anodic peaks observed are due to the presence of the lysine residues, however, the CV scan of the MOG  peptide exhibits a higher current and a wider electrochemical window compared to the CV scan of MOG 37-55 peptide, as the latter contained only 1 lysine residue.
DPV is a more sensitive approach compared to CV and hence, has been extensively used as a more sensitive method for the detection of molecules in low concentration [38]. In Figure 5, the DPVs are recorded for the bare film electrode, as well as for each part that constitutes the final structure of OM-(KG) 5 -MOG 35-55 conjugate on the Graphite/SiO 2 working electrode. As can be seen in Figure 5a, the bare graphite/SiO 2 is free of any redox peaks. However, in Figure 5b, there are two peaks which correspond to (KG) 5 -MOG  peptide, approximately at −0.65 V and −0.27 V, respectively. Figure 5c shows the DPV of mannan (in 0.1 M phosphate buffer) on the surface of the film electrode, displaying a clear sharper peak at around −0.26 V. The last step in order to evaluate the conjugation of peptide (KG) 5 -MOG  with OM via DPV measurements is depicted in Figure 5d with a clear and distinct peak at −0.28 V and a shoulder peak at −0.62 V, which are actually due to the presence of OM-(KG) 5 -MOG  Brain Sci. 2020, 10, 577 8 of 13 on the graphite/SiO 2 film electrode (after the addition of 0.002 mg/mL of OM). This is a proof of concept study, and we intend to further study the quantification of this and other conjugates, focusing on the limit od detection (LOD) of these conjugates using voltammetric techniques.  DPV is a more sensitive approach compared to CV and hence, has been extensively used as a more sensitive method for the detection of molecules in low concentration [38]. In Figure 5, the DPVs are recorded for the bare film electrode, as well as for each part that constitutes the final structure of OM-(KG)5-MOG35-55 conjugate on the Graphite/SiO2 working electrode. As can be seen in Figure 5a, the bare graphite/SiO2 is free of any redox peaks. However, in Figure 5b, there are two peaks which correspond to (KG)5-MOG35-55 peptide, approximately at −0.65 V and −0.27 V, respectively. Figure 5c shows the DPV of mannan (in 0.1 M phosphate buffer) on the surface of the film electrode, displaying a clear sharper peak at around −0.26 V. The last step in order to evaluate the conjugation of peptide (KG)5-MOG35-55 with OM via DPV measurements is depicted in Figure 5d with a clear and distinct peak at −0.28 V and a shoulder peak at −0.62 V, which are actually due to the presence of OM-(KG)5-MOG35-55 on the graphite/SiO2 film electrode (after the addition of 0.002 mg/mL of OM). This is a proof of concept study, and we intend to further study the quantification of this and other conjugates, focusing on the limit od detection (LOD) of these conjugates using voltammetric techniques.   DPV is a more sensitive approach compared to CV and hence, has been extensively used as a more sensitive method for the detection of molecules in low concentration [38]. In Figure 5, the DPVs are recorded for the bare film electrode, as well as for each part that constitutes the final structure of OM-(KG)5-MOG35-55 conjugate on the Graphite/SiO2 working electrode. As can be seen in Figure 5a, the bare graphite/SiO2 is free of any redox peaks. However, in Figure 5b, there are two peaks which correspond to (KG)5-MOG35-55 peptide, approximately at −0.65 V and −0.27 V, respectively. Figure 5c shows the DPV of mannan (in 0.1 M phosphate buffer) on the surface of the film electrode, displaying a clear sharper peak at around −0.26 V. The last step in order to evaluate the conjugation of peptide (KG)5-MOG35-55 with OM via DPV measurements is depicted in Figure 5d with a clear and distinct peak at −0.28 V and a shoulder peak at −0.62 V, which are actually due to the presence of OM-(KG)5-MOG35-55 on the graphite/SiO2 film electrode (after the addition of 0.002 mg/mL of OM). This is a proof of concept study, and we intend to further study the quantification of this and other conjugates, focusing on the limit od detection (LOD) of these conjugates using voltammetric techniques.

Complete Conjugation between (KG) 5 -MOG 35-55 Peptide to OM is Monitored by HPLC
Contrarily to the conjugation of MOG 35-55 peptide with mannan, which did not occur, the reaction of (KG) 5 -MOG 35-55 with mannan (oxidized or not) resulted in gradual conjugation of (KG) 5 -MOG  peptide within 6 h depicted in the gradual loss of the HPLC peak during this period ( Figure 6). The amino groups of lysine residues within (KG) 5 forms a Schiff base reaction with the aldehyde groups of OM (resulting after the oxidation of mannan). The (KG) 5 -MOG  peptide peak at 9.62 gradually disappears within this period, showing complete conjugation of (KG) 5 -MOG  peptide to OM. Figure 6b shows the completion of conjugation within six hours. of (KG)5-MOG35-55 with mannan (oxidized or not) resulted in gradual conjugation of (KG)5-MOG35-55 peptide within 6 h depicted in the gradual loss of the HPLC peak during this period ( Figure 6). The amino groups of lysine residues within (KG)5 forms a Schiff base reaction with the aldehyde groups of OM (resulting after the oxidation of mannan). The (KG)5-MOG35-55 peptide peak at 9.62 gradually disappears within this period, showing complete conjugation of (KG)5-MOG35-55 peptide to OM. Figure 6b shows the completion of conjugation within six hours.

The Importance of the Linker (KG)5 for Conjugation of Peptides to OM
The conjugation of MOG35-55 peptide to OM was achieved through (KG)5 linker, as previously described [23]. As demonstrated, this approach provides simple and efficient conjugation by the Schiff base reaction, where aldehyde groups of OM reacts with the amino groups of the lysine side chains of the (KG)5-MOG35-55, peptide. In previous similar studies using the linker KG of varying lengths, (KG)n=1-5, we noted that the length of the linker plays a crucial role in the ability of peptides to be efficiently conjugated to the OM scaffold [48].

Mannan-Peptide Conjugate
In the OM-(KG)5-MOG35-55 conjugate, unreacted aldehyde groups are necessary to immunoregulate the peptide to dendritic cells. This is a result of ethylene glycol addition to blockade further oxidation, and in line with previous studies on MUC1-mannan conjugates in cancer research, which required aldehyde groups in order to activate dendritic cells [39]. The matrix also contains intact mannose units, not oxidized, necessary to bind to the mannose receptor of the dendritic cells and their activation via toll-like receptor 4 [49][50][51][52]. In particular, the procedure we followed to produce the mannan-peptide conjugate allows: (i) the presence of antigen peptide MOG35-55 3.5. The Importance of the Linker (KG) 5

for Conjugation of Peptides to OM
The conjugation of MOG  peptide to OM was achieved through (KG) 5 linker, as previously described [23]. As demonstrated, this approach provides simple and efficient conjugation by the Schiff base reaction, where aldehyde groups of OM reacts with the amino groups of the lysine side chains of the (KG) 5 -MOG  , peptide. In previous similar studies using the linker KG of varying lengths, (KG) n=1-5 , we noted that the length of the linker plays a crucial role in the ability of peptides to be efficiently conjugated to the OM scaffold [48].

Mannan-Peptide Conjugate
In the OM-(KG) 5 -MOG 35-55 conjugate, unreacted aldehyde groups are necessary to immunoregulate the peptide to dendritic cells. This is a result of ethylene glycol addition to blockade further oxidation, and in line with previous studies on MUC1-mannan conjugates in cancer research, which required aldehyde groups in order to activate dendritic cells [39]. The matrix also contains intact mannose units, not oxidized, necessary to bind to the mannose receptor of the dendritic cells and their activation via toll-like receptor

Chemistry of the Mannose Cleavage
The cis-diols can form a cyclic complex upon oxidation with strong oxidizing agents as periodate. This allows the cleavage of the bond between the two carbons bearing the two hydroxyl groups, leading to the formation of aldehyde groups. Mannose is a carbohydrate, which holds two hydroxyl groups at positions 2,3 of the ring in a cis-position. This allows the oxidizing agent sodium periodate to form a cyclic complex, which finally leads to cleavage of the carbon-carbon bond bearing the cis-hydroxyl groups. This complex cannot be formed if the hydroxyl groups at the adjacent carbon atoms are in a trans position and subsequently this carbon-carbon bond cannot be cleaved. The formation of the cyclic mannose-periodate complex is leading finally to the cleavage of the ring and the formation of the two aldehyde groups. These groups react with the amino groups of the five lysines of the (KG) 5 -MOG  to form double bond imines (Schiff base reaction) thus, the MOG 35-55 peptide attached to the mannan scaffold. Figure 7 shows the mechanism of cis diol cleavage.
Brain Sci. 2020, 10, x FOR PEER REVIEW 10 of 14 connected with aldehyde groups of the OM through immune bonds (Schiff base) with the amino groups of the lysine side chain in the (KG)5-MOG35-55 peptide. The peptide-OM conjugate is delivered to dendritic cells via the mannan scaffold for regulation of the immune system; (ii) the presence of unreacted aldehyde groups are necessary to modulate dendritic cells; and (iii) the presence intact mannose units, not oxidized, necessary to bind to the mannose receptor of the dendritic cells.

Chemistry of the Mannose Cleavage
The cis-diols can form a cyclic complex upon oxidation with strong oxidizing agents as periodate. This allows the cleavage of the bond between the two carbons bearing the two hydroxyl groups, leading to the formation of aldehyde groups. Mannose is a carbohydrate, which holds two hydroxyl groups at positions 2,3 of the ring in a cis-position. This allows the oxidizing agent sodium periodate to form a cyclic complex, which finally leads to cleavage of the carbon-carbon bond bearing the cis-hydroxyl groups. This complex cannot be formed if the hydroxyl groups at the adjacent carbon atoms are in a trans position and subsequently this carbon-carbon bond cannot be cleaved. The formation of the cyclic mannose-periodate complex is leading finally to the cleavage of the ring and the formation of the two aldehyde groups. These groups react with the amino groups of the five lysines of the (KG)5-MOG35-55 to form double bond imines (Schiff base reaction) thus, the MOG35-55 peptide attached to the mannan scaffold. Figure 7 shows the mechanism of cis diol cleavage.

Conclusions
We developed and confirm an analytical electrochemical method for monitoring the conjugation reaction of peptides to the carrier mannan; (KG)5-MOG35-55 was used as the peptide example in this study. Peptide-OM conjugates can serve as potential vaccine candidates as has previously been shown by the group for cancer models and more recently in MS models. Electrochemical voltammetric techniques and HPLC experiments were used to confirm the conjugation of (KG)5-MOG35-55 to the aldehyde groups of OM. It is shown that voltammetric technique and HPLC can be used to monitor the conjugation efficiency of peptide-carrier conjugates.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: EDS elemental microanalysis of a Graphite/SiO2 film electrode.

Conclusions
We developed and confirm an analytical electrochemical method for monitoring the conjugation reaction of peptides to the carrier mannan; (KG) 5 -MOG  was used as the peptide example in this study. Peptide-OM conjugates can serve as potential vaccine candidates as has previously been shown by the group for cancer models and more recently in MS models. Electrochemical voltammetric techniques and HPLC experiments were used to confirm the conjugation of (KG) 5 -MOG  to the aldehyde groups of OM. It is shown that voltammetric technique and HPLC can be used to monitor the conjugation efficiency of peptide-carrier conjugates.