Name: activation and conjugation of soluble polysaccharides using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (cdap)
Uploaded: 2021-06-14T13:00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP at JoVE.com

The work described here was funded by Fina Biosolutions LLC.

<ol>
	<li>Ellis, R. W., Granoff, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development and clinical uses of Haemophilus B conjugate vaccines. , (1994).</li><li>Goebel, W. F., Avery, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemo-immunological+studies+on+conjugated+carbohydrate-proteins.">Chemo-immunological studies on conjugated carbohydrate-proteins.</a> Journal of Experimental Medicine. 50 (4), 533-550 (1929).</li><li>Mond, J. J., Vos, Q., Lees, A., Snapper, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+independent+antigens.">T cell independent antigens.</a> Current Opinion in Immunology. 7 (3), 349-354 (1995).</li><li>Cruse, J. M., Lewis, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conjugate Vaccines. 10, (1989).</li><li>Lees, A., Nelson, B. L., Mond, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents.</a> Vaccine. 14 (3), 190-198 (1996).</li><li>Shafer, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-4-dimethylaminopyridinium+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+II.+Selective+crosslinking+of+proteins+to+CDAP-activated+polysaccharides.">Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides.</a> Vaccine. 18 (13), 1273-1281 (2000).</li><li>Schneerson, R., Barrera, O., Sutton, A., Robbins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+immunogenicity+of+Haemophilus+influenzae+type+b+polysaccharide-protein+conjugates.">Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates.</a> Journal of Experimental Medicine. 152 (2), 361-376 (1980).</li><li>Lees, A., Barr, J. F., Gebretnsae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+soluble+polysaccharides+with+1-cyano-+4-dimethylaminopyridine+tetrafluoroborate+(CDAP)+for+use+in+protein-polysaccharide+conjugate+vaccines+and+immunological+reagents.+III+Optimization+of+CDAP+activation.">Activation of soluble polysaccharides with 1-cyano- 4-dimethylaminopyridine tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. III Optimization of CDAP activation.</a> Vaccines. 8 (4), 777 (2020).</li><li>Qi, X. -. Y., Keyhani, N. O., Lee, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+hydrazine,+hydrazides,+and+their+mixtures+with+trinitrobenzenesulfonic+acid.">Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid.</a> Analytical biochemistry. 175 (1), 139-144 (1988).</li><li>Monsigny, M., Petit, C., Roche, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+determination+of+neutral+sugars+by+a+resorcinol+sulfuric+acid+micromethod.">Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod.</a> Analytical biochemistry. 175 (2), 525-530 (1988).</li><li>Hermanson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. 3rd ed. , (2013).</li></ol>

Andrew Lees is founder and owner of Fina Biosolutions. He holds several patents relating to CDAP chemistry and benefits from licensing of the chemistry and CDAP conjugation know-how.

CDAP is a convenient reagent to derivatize and conjugate polysaccharides. This article describes the general method to use CDAP to derivatize polysaccharides with hydrazides (PS-ADH) and incorporates recently published improvements8. First, the technique emphasizes the importance of maintaining the target pH to control the activation process. We found that while many common buffers interfere with the CDAP activation reaction, DMAP could successfully be used as the buffer to manage the pH8. Furthermore, DMAP is already a reaction byproduct of CDAP activation. Finally, buffering the polysaccharide solution with DMAP before adding the CDAP facilitates precise targeting and maintenance of the reaction pH. As we describe, it is useful to adjust the pH of the concentrated DMAP stock solution such that when diluted, it reaches the targeted pH. Secondly, performing the process in the cold slowed the reaction time, making the activation process less frenetic and more forgiving. Lower temperature decreased the rate of CDAP hydrolysis, and the optimal activation time at pH 9 increases from ~3 min to ~15 min. In addition, less CDAP is required to achieve the same level of activation than when performed at room temperature. 
ADH-derivatized polysaccharides can be conjugated to proteins using carbodiimides (e.g., EDAC)7. For example, several licensed Haemophilus influenzae b (Hib) vaccines use the polyribosylribitolphosphate (PRP) derivatized with ADH to conjugate to tetanus toxoid using EDAC. CNBr was initially employed, but CDAP is a much easier reagent to use for this purpose. In our experience, a good target range for ADH derivatization is 10-30 hydrazides per 100 kDa polysaccharide or ~1-3% ADH by weight. 
The same process can be used to derivatize polysaccharides with primary amines by substituting the ADH for a diamine. It is recommended to use hexane diamine to derivatize polysaccharides with amines8. The aminated polysaccharide (PS-NH2) can be conjugated using reagents developed for protein conjugation11. Typically, the PS-NH2 is derivatized with a maleimide (e.g., succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) or N-&#947;-maleimidobutyryl-oxysuccinimide ester (GMBS)), and the protein is thiolated (e.g., with succinimidyl 3-(2-pyridyldithio)propionate (SPDP)). Thiol-maleimide chemistry is very efficient.
Proteins can also be directly coupled to CDAP-activated polysaccharides via the &#603;-amine on lysines. While the activation protocol used is generally similar to the one described here, it is necessary to optimize the level of activation, polysaccharide and protein concentration, as well as the protein:polysaccharide ratio5,6,8. 
Dextran is one of the easiest polysaccharides to activate with CDAP due to its relatively high density of hydroxyl groups, but some polysaccharides, such as Vi antigen, can be challenging. Consequently, there is no single "best" protocol for CDAP conjugation directly to proteins. We suggest first developing a protocol to achieve suitable levels of activation, as determined by the extent of hydrazide derivatization, and then proceeding to direct protein conjugation to CDAP-activated polysaccharide.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.
Figure 4 was updated from:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig04.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62597/activation-conjugation-soluble-polysaccharides-using-1-cyano-4">Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)</a>. A figure&#160;was updated.

Erratum: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines, such as those consisting of polysaccharides covalently linked to a carrier protein, are among the remarkable advances in vaccinology1,2. Polysaccharides, as T-cell-independent antigens, are poorly immunogenic in infants and do not induce memory, class switching, or affinity maturation of antibodies3. These shortcomings are overcome in polysaccharide conjugate vaccines4. As most polysaccharides do not have a convenient chemical handle for conjugation, they must first be made reactive or &#34;activated.&#34; The activated polysaccharide is then linked either directly with the protein (or modified protein) or is functionalized for additional derivatization before conjugation4. Most licensed polysaccharide conjugate vaccines use either reductive amination or cyanylation to activate polysaccharide hydroxyls. Cyanogen bromide (CNBr), a reagent that had previously been used to activate chromatography resins, was initially used for polysaccharide derivatization. However, CNBr requires high pH, typically ~ pH 10.5 or greater, to partially deprotonate polysaccharide hydroxyls so that they are sufficiently nucleophilic to attack the cyano group. The high pH can be detrimental to base-labile polysaccharides, and neither CNBr nor the active cyano-ester initially formed is sufficiently stable at such high pH.
CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate; Figure 1) was introduced by Lees et al. for use as a cyanylating agent for the activation of polysaccharides5,6. CDAP, which is crystalline and easy to handle, was found to activate polysaccharides at a lower pH than CNBr and with fewer side reactions. Unlike CNBr, CDAP-activated polysaccharides can be directly conjugated to proteins, simplifying the synthesis process. CDAP-activated polysaccharides can be functionalized with a diamine (e.g., hexane diamine) or a dihydrazide (e.g., adipic dihydrazide, ADH) to make amino- or hydrazide-derivatized polysaccharides. A high concentration of the homobifunctional reagent is used to suppress crosslinking of polysaccharides. Amino polysaccharides can then be conjugated using any of the myriad techniques used for protein conjugation. Hydrazide-derivatized polysaccharides are often coupled to proteins using a carbodiimide reagent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC))7. Further optimization of CDAP polysaccharide activation has been described by Lees et al.8 and is incorporated into the protocol described here.
CDAP conjugation overview 
The CDAP protocol can be conceptualized as two phases: (1) the activation of the polysaccharide and (2) conjugation of the activated polysaccharide with a protein or ligand (Figure 2). The goal of the first step is to efficiently activate the polysaccharide, while the goal of the second is to efficiently conjugate to the activated polysaccharide. The activated polysaccharide ties the two steps together. This conceptualization helps focus on the critical elements of each step. Figure 2 expands on this conceptualization, showing the desired activation and coupling reactions, along with the hydrolysis reactions and side reactions.
During the activation phase, the three major concerns are CDAP stability, CDAP reaction with the polysaccharide hydroxyls, and the stability of the activated polysaccharide (Figure 3). CDAP hydrolysis increases with pH, as does the hydrolysis of the activated polysaccharide and the side reactions. However, the CDAP reaction with the polysaccharide is facilitated by increasing the pH. Efficiently activating polysaccharides with CDAP requires a balance between 1) the reactivity of the polysaccharide and CDAP and 2) the hydrolysis and side reactions of both the reagent and the activated polysaccharide.
In the original CDAP activation protocol described by Lees et al.5, CDAP activation of polysaccharides was carried out at room temperature in unbuffered pH 9 solution. The rate of activation was found to be rapid under this condition, and the activation would be complete within 3 min. The reaction was also accompanied by rapid hydrolysis of CDAP, causing a rapid pH drop of the unbuffered reaction solution. It was challenging to quickly raise and to maintain the reaction pH at the target value in such a short time frame. In the described protocol, activation was performed by adding CDAP from a 100 mg/mL stock solution to the unbuffered polysaccharide solution. The pH was raised 30 s later with &#34;an equal volume of 0.2 M triethylamine&#34;. Protein to be conjugated was then added after 2.5 min to the activation reaction. Notably, the pH of the activation step was not well controlled and most likely initially exceeded the target pH. The fast reaction requiring prompt pH adjustment made the activation process difficult to control and challenging to scale up.
In contrast to the original protocol, the modified protocol described here has two major improvements. First, the pH of the polysaccharide solution is pre-adjusted to target activation pH, using DMAP as the buffer, before the addition of CDAP. DMAP has a pKa of 9.5 and thus has good buffering power around pH 9, and unlike many other buffers, DMAP was not found to promote CDAP hydrolysis8. Furthermore, DMAP is already a process intermediate and therefore does not add a new component to the reaction mixture. Pre-adjusting the pH before adding CDAP eliminates the large pH swing at the beginning of the reaction and allows for more efficient maintenance of the target pH during the reaction. The second improvement is to perform the activation reaction at 0 &#176;C, where the rate of CDAP hydrolysis is markedly slower than that at room temperature. With the longer reagent half-life at 0 &#176;C, the activation time is increased from 3 min to 15 min to compensate for the slower activation rate at the lower temperature. The longer reaction time, in turn, makes it easier to maintain the reaction pH. The use of 0 &#176;C also slows the degradation of pH-sensitive polysaccharides, making it possible to prepare conjugates of this type of polysaccharide. The improvements in the protocol make the activation process less frenetic, easier to control, more reproducible, and more amenable to scaling up.
This article describes the improved protocol for carrying out controlled CDAP activation of polysaccharide at 0 &#176;C and at a specified target pH and performing subsequent derivatization of the activated polysaccharides with ADH. Also described is a trinitrobenzene sulfonic acid (TNBS) assay, based on the method of Qi et al.9, for the determination of hydrazide level on the modified polysaccharide. A modified assay for hexoses based on resorcinol and sulfuric acid10 is also described, which can be used for determining a broader range of polysaccharides. For more information on CDAP activation and conjugation, the reader is referred to earlier publications5,6,8 by Lees et al.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid dihydrazide</td>
			<td>Sigma</td>
			<td>A0638</td>
			<td>MW 174</td>
		</tr>
		<tr>
			<td>Amicon Ultra 15 10 kDa</td>
			<td>Millipore</td>
			<td>UFC901008</td>
			<td>MW cutoff can be 30 kDa for 200 kDa PS</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autotitrator or electronic pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker&#160; 2-4 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CDAP</td>
			<td>SAFC</td>
			<td>RES1458C</td>
			<td>Sigma</td>
		</tr>
		<tr>
			<td>DMAP</td>
			<td>Sigma</td>
			<td>107700</td>
			<td>MW 122.2</td>
		</tr>
		<tr>
			<td>Flake ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl 1 M</td>
			<td>VWR</td>
			<td>BDH7202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro stir bar</td>
			<td>VWR</td>
			<td>76001-878</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tube (for CDAP)</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>BDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 1 M</td>
			<td>Sigma</td>
			<td>1099130001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 10 M</td>
			<td>Sigma</td>
			<td>SX0607N-6</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td>Cole Parmer</td>
			<td>55510-22</td>
			<td>6 mm x 110 mm Epoxy single junction</td>
		</tr>
		<tr>
			<td>pH temperature probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipets &#38; tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saline or PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small beaker 5-20 mL</td>
			<td>VWR</td>
			<td>10754-696</td>
			<td>A 10 mL beaker allows room for pH probe &#38; pipet</td>
		</tr>
		<tr>
			<td>Small ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcinol assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Combitip</td>
			<td>Eppendorf</td>
			<td>10 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Repligen</td>
			<td>132650T</td>
			<td>Spectra/Por 6-8kDa</td>
		</tr>
		<tr>
			<td>Dialysis tubing clips</td>
			<td>Repligen</td>
			<td>142150</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeat pipettor</td>
			<td>Eppendorf</td>
			<td>M4</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcinol</td>
			<td>Sigma</td>
			<td>398047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sugar standard</td>
			<td></td>
			<td>As appropriate</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sulfuric acid 75%</td>
			<td>VWR</td>
			<td>BT126355-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNBS assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic dihydrazide</td>
			<td>Sigma</td>
			<td>A0638</td>
			<td>MW 174</td>
		</tr>
		<tr>
			<td>Borosilcate test tubes 12 x 75</td>
			<td>VWR</td>
			<td>47729-570</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borate, 0.5 M pH 9</td>
			<td>Boston Biologicals</td>
			<td>BB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>TNBS 5% w/v</td>
			<td>Sigma</td>
			<td>P2297</td>
			<td>MW 293.17</td>
		</tr>
	</tbody>
</table>

activation and conjugation of soluble polysaccharides using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (cdap)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be conveniently functionalized and linked to vaccine carrier proteins using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), an easy-to-handle cyanylating reagent. CDAP activates polysaccharides by reacting with carbohydrate hydroxyl groups at pH 7-9. The stability and reactivity of CDAP are highly pH-dependent. The pH of the reaction also decreases during activation due to the hydrolysis of CDAP, which makes good pH control the key to reproducible activation. The original CDAP activation protocol was performed at room temperature in unbuffered pH 9 solutions.
Due to the rapid reaction under this condition (&#60;3 min) and the accompanying fast pH drop from the rapid CDAP hydrolysis, it was challenging to quickly adjust and maintain the target reaction pH in the short time frame. The improved protocol described here is performed at 0 &#176;C, which slows CDAP hydrolysis and extends the activation time from 3 min to ~15 min. Dimethylaminopyridine (DMAP) was also used as a buffer to pre-adjust the polysaccharide solution to the target activation pH before adding the CDAP reagent. The longer reaction time, coupled with the slower CDAP hydrolysis and the use of DMAP buffer, makes it easier to maintain the activation pH for the entire duration of the activation process. The improved protocol makes the activation process less frenetic, more reproducible, and more amenable to scaling up.

To illustrate the activation and derivatization of a polysaccharide using CDAP chemistry, dextran was activated at 0.25 and 0.5 mg CDAP/mg dextran. For each reaction, a 10 mg/mL dextran solution in water was chilled on ice, and 1/10th volume of a 2.5 M DMAP stock (prepared as described in section 3) was added. The final solution was brought to pH 9 by the addition of 0.1 M NaOH in 10 &#181;L aliquots. The solution was chilled and stirred, CDAP added, and the pH maintained at pH 9 by adding 10 &#181;L aliquots of 0.1 M NaOH for 15 min. Only 0.25 mL of 0.5 M ADH at pH 9 was added (less than the usual amount) and the reaction allowed to proceed overnight at 4 &#176;C.
The labeled dextran was then sequentially dialyzed against 1 M NaCl, 0.15 M NaCl, and water as described in section 6. The ADH-dextran was then assayed for dextran using the resorcinol/sulfuric acid assay (section 7.2). A typical standard curve using glucose as the sugar standard is shown in Figure 4A. The hydrazide content was determined using the TNBS assay described in section 7.3. A typical hydrazide standard curve using ADH as the standard is given in Figure 4B.
Representative calculations from the activation of dextran at the two levels of activation are shown in Figure 4A,B. The data are presented as both hydrazides per 100 kDa of dextran polymer and as a weight percent of ADH to dextran, as described in sections 7.9.3.4 and 7.9.3.5, respectively, in Figure 4C. The degree of derivatization approximately doubled as the CDAP ratio was doubled.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62597/62597fig01.jpg" /> 
Figure 1: Chemical structure of CDAP. CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62597/62597fig02.jpg" /> 
Figure 2: Process of CDAP activation and conjugation. The process is conceptually divided into two phases, with the activated polysaccharide common to both. Under basic conditions, CDAP activates polysaccharide hydroxyls, releasing DMAP (reaction 1). CDAP hydrolysis also releases DMAP (reaction 3). Although a cyano-ester is shown, this may not be the actual intermediate. The intermediate is, therefore, referred to as (CDAP) &#34;activated&#34; polysaccharide. During the first activation phase, the activated polysaccharide can hydrolyze (reaction 4) or undergo side reactions (reaction 5). In the second conjugation phase (reaction 2), the activated polysaccharide reacts with an amine to form a stable isourea bond in addition to reactions 4 and 5. Abbreviations: CDAP = 1-cyano-4-dimethylaminopyridine tetrafluoroborate; DMAP = 4-dimethylaminopyridine; R-NH2 = amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62597/62597fig03.jpg" /> 
Figure 3: CDAP activation and conjugation. The process requires balancing CDAP reactivity with the polysaccharide, the stability of the CDAP and activated polysaccharide, as well as the reactivity of the activated polysaccharide with that of the amine. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62597/62597fig4v4.jpg" /> 
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as &#181;M Hz.&#160;(C)&#160;Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. <a href="https://www.jove.com/files/ftp_upload/62597/62597fig4v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP at JoVE.com

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 &#176;C and varying pH and performing subsequent conjugation of the activated polysaccharides.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

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