We would like to thank A. Resemann, F. J. Mayer, and D. Suckau from Bruker Daltonics GmbH Bremen; D. Tietze, A. A. Tietze, V. Schmidts and C. Thiele from the Darmstadt University of Technology; O. Ohlenschl&#228;ger from the FLI Jena, M. Engeser from the University of Bonn; K. Kramer, A. Harzen, and H. Nakagami from the Max Planck Institute for Plant Breeding Research, Cologne; Susanne Neupert from the Institute for Zoology, Cologne; and the Biomolecular Magnetic Resonance Spectroscopy Facilities of the University of Frankfurt for technical support, training modules, and access to instruments. Financial support by the University of Bonn to D.I. is gratefully acknowledged.

Note: All amino acids used herein were in the L-configuration. The abbreviations of amino acids and amino acid derivatives were used according to the recommendations of the Nomenclature Committee of IUB and the IUPAC-IUB Joint Commission on Biochemical Nomenclature.
1. Solid-Phase Peptide Synthesis (SPPS)
NOTE: Carry out the synthesis with a solid-phase peptide synthesizer. Perform the synthesis of the linear peptide precursors of the general sequence ZRLCCGFOKSCRSRQC...

<ol>
	<li>Fosgerau, K., Hoffmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+therapeutics:+Current+status+and+future+directions.">Peptide therapeutics: Current status and future directions.</a> Drug Discovery Today. 20 (1), 122-128 (2015).</li><li>Gongora-Ben&#237;tez, M., Tulla-Puche, J., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+roles+of+disulfide+bonds.+peptides+as+therapeutics.">Multifaceted roles of disulfide bonds. peptides as therapeutics.</a> Chemical Reviews. 114 (2), 901-926 (2014).</li><li>Tietze, A. A., 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=Structurally+diverse+&#181;-conotoxin+PIIIA+isomers+block+sodium+channel+NaV1.4.">Structurally diverse &#181;-conotoxin PIIIA isomers block sodium channel NaV1.4.</a> Angewandte Chemie - International Edition. 51 (17), 4058-4061 (2012).</li><li>Carstens, B. B., 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=Structure-Activity+Studies+of+Cysteine-Rich+&#945;-Conotoxins+that+Inhibit+High-Voltage-Activated+Calcium+Channels+via+GABABReceptor+Activation+Reveal+a+Minimal+Functional+Motif.">Structure-Activity Studies of Cysteine-Rich &#945;-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABABReceptor Activation Reveal a Minimal Functional Motif.</a> Angewandte Chemie - International Edition. 55 (15), 4692-4696 (2016).</li><li>Zhang, Y., Schulten, K., Gruebele, M., Bansal, P. S., Wilson, D., Daly, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bridges:+Bringing+together+frustrated+structure+in+a+bioactive+peptide.">Disulfide bridges: Bringing together frustrated structure in a bioactive peptide.</a> Biophysical Journal. 110 (8), 1744-1752 (2016).</li><li>Nielsen, K. J., 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=Solution+structure+of+&#181;-conotoxin+PIIIA,+a+preferential+inhibitor+of+persistent+tetrodotoxin-sensitive+sodium+channels.">Solution structure of &#181;-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels.</a> Journal of Biological Chemistry. 277 (30), 27247-27255 (2002).</li><li>Heimer, P., 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=Conformational+&#181;-Conotoxin+PIIIA+Isomers+Revisited:+Impact+of+Cysteine+Pairing+on+Disulfide-Bond+Assignment+and+Structure+Elucidation.">Conformational &#181;-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.</a> Analytical Chemistry. 90 (5), 3321-3327 (2018).</li><li>Chang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+pathways+of+oxidative+folding+of+disulfide+proteins:+Underlying+causes+and+folding+models.">Diverse pathways of oxidative folding of disulfide proteins: Underlying causes and folding models.</a> Biochemistry. 50 (17), 3414-3431 (2011).</li><li>B&#246;hm, M., 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=Novel+insights+into+structure+and+function+of+factor+XIIIa-inhibitor+tridegin.">Novel insights into structure and function of factor XIIIa-inhibitor tridegin.</a> Journal of Medicinal Chemistry. 57 (24), 10355-10365 (2014).</li><li>Postma, T. M., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+Formation+Strategies+in+Peptide+Synthesis.">Disulfide Formation Strategies in Peptide Synthesis.</a> European Journal of Organic Chemistry. 2014 (17), 3519-3530 (2014).</li><li>Albericio, F., Isidro-llobet, A., Mercedes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+Acid-Protecting+Groups.">Amino Acid-Protecting Groups.</a> Chemical Reviews. 109 (6), 2455-2504 (2009).</li><li>Annis, I., Hargittai, B., Barany, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bond+formation+in+peptides.">Disulfide bond formation in peptides.</a> Methods in Enzymology. 289 (1988), 198-221 (1997).</li><li>Kamber, B., 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=The+Synthesis+of+Cystine+Peptides+by+Iodine+Oxidation+of+S-Trityl-cysteine+and+S-Acetamidomethyl-cysteine+Peptides.">The Synthesis of Cystine Peptides by Iodine Oxidation of S-Trityl-cysteine and S-Acetamidomethyl-cysteine Peptides.</a> Helvetica Chimica Acta. 63 (4), 899-915 (1980).</li><li>Bosch, D. E., Zielinski, T., Lowery, R. G., Siderovski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Modulators+of+Regulator+of+G-Protein+Signaling+(RGS)+Proteins.">Evaluating Modulators of Regulator of G-Protein Signaling (RGS) Proteins.</a> Current Protocols in Pharmacology. 56 (2.8), 1-15 (2012).</li><li>Mochizuki, M., Tsuda, S., Tanimura, K., Nishiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regioselective+formation+of+multiple+disulfide+bonds+with+the+aid+of+postsynthetic+S-tritylation.">Regioselective formation of multiple disulfide bonds with the aid of postsynthetic S-tritylation.</a> Organic Letters. 17 (9), 2202-2205 (2015).</li><li>Peigneur, S., 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=&#948;-conotoxins+synthesized+using+an+acid-cleavable+solubility+tag+approach+reveal+key+structural+determinants+for+NaV+subtype+selectivity.">&#948;-conotoxins synthesized using an acid-cleavable solubility tag approach reveal key structural determinants for NaV subtype selectivity.</a> Journal of Biological Chemistry. 289 (51), 35341-35350 (2014).</li><li>Heimer, P., 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=Application+of+Room-Temperature+Aprotic+and+Protic+Ionic+Liquids+for+Oxidative+Folding+of+Cysteine-Rich+Peptides.">Application of Room-Temperature Aprotic and Protic Ionic Liquids for Oxidative Folding of Cysteine-Rich Peptides.</a> ChemBioChem. 15 (18), 2754-2765 (2014).</li><li>Kates, S. A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid-Phase Synthesis: A Practical Guide. , (2000).</li><li>W&#252;thrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+structure+and+function+of+biological+macromolecules+(Nobel+lecture).">NMR studies of structure and function of biological macromolecules (Nobel lecture).</a> Angewandte Chemie - International Edition. 42 (29), 3340-3363 (2003).</li></ol>

The authors have nothing to disclose.

The method described herein for the synthesis of cysteine-rich peptides such as &#181;-PIIIA represents a possibility to selectively produce disulfide-bonded isomers from the same amino acid sequence. Therefore, established methods such as Fmoc-based solid phase peptide synthesis18 and a defined protecting group strategy for the regioselective formation of disulfide bonds were used16. The solid-phase peptide synthesis can produce amino acid sequences on a polymer support (r...

An erratum was issued for: <a href="https://www.jove.com/video/58368/synthesis-structure-determination-conotoxin-piiia-isomers-with">Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities</a>. The Protocol and Materials sections were updated.
Step 1.1.3 in the Protocol section was updated from:
Weigh the individual reagents (amino acids, HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)) according to the protocol and dissolve them in dimethylformamide (DMF) to a final concentration of 2.4 M (amino acids) and 0.6 M (HBTU), respectively.
to:
Weigh the individual reagents (amino acids, HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)) according to the protocol and dissolve them in dimethylformamide (DMF) to a final concentration of 0.6 M (amino acids) and 0.6 M (HBTU), respectively.
Step 1.2 in the Protocol section was updated from:
NOTE: The protocol applies to 100 mg of resin (loading: 0.28 mmol/g) added to one reaction column for 28 &#956;mol scale.
to:
NOTE: The standardized protocol usually applies to 100 mg of resin (loading: 0.53 mmol/g) added to one reaction column for 53 &#956;mol scale leading to the following equivalents: 5 eq. HBTU, 10 eq. NMM, 5 eq. amino acid. In case of PIIIA, however, a loading of 0.28 mmol/g (28 &#181;mol scale) is used, which results in the specified higher equivalents.
Step 1.2.3.1 in the Protocol section was updated from:
HBTU (450 &#181;L; 0.6 M in DMF; 270 &#181;mol; 9.6 eq.), NMM (125 &#181;L; 50% in DMF; 568 &#181;mol; 20 eq.), Fmoc-amino acid (420 &#181;L; 2.4 M in DMF; 1.01 mmol; 36 eq.).
to:
HBTU (415 &#181;L; 0.6 M in DMF; 249 &#181;mol; 9 eq.), NMM (112 &#181;L; 50% in DMF; 510 &#181;mol; 18 eq.), Fmoc-amino acid (420 &#181;L; 0.6 M in DMF; 252 &#181;mol; 9 eq.).
The first item in the Materials table was updated from:
<table>
	<tbody>
		<tr>
			<td>Fmoc Rink amide resin</td>
			<td>Novabiochem</td>
			<td>855001</td>
			<td></td>
		</tr>
	</tbody>
</table>
to:
<table>
	<tbody>
		<tr>
			<td>PS PEG2000 Fmoc Rink-amide Resin</td>
			<td>Varian, Inc.</td>
			<td>PL3867-3764</td>
			<td>AmphiSpheres 40 RAM</td>
		</tr>
	</tbody>
</table>

An erratum was issued for: <a href="https://www.jove.com/video/58368/synthesis-structure-determination-conotoxin-piiia-isomers-with">Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities</a>. The Protocol and Materials sections were updated.

Erratum: Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

The use of bioactive peptides in pharmaceutical research and development is highly recognized, because they represent potent and highly selective compounds for specific biological targets1. For their bioactivity, however, the three-dimensional structure is of great importance in order to perform structure-activity relationship studies2,3,4. Apart from the primary amino acid sequence that influences the overall conformation, disulfide bonds significantly stabilize the structure of cysteine-rich peptides5. Multiple disulfide-bridged peptides include conotoxins such as &#181;-PIIIA from Conus purpurascens which contains six cysteines in its sequence. This high cysteine content theoretically allows the formation of 15 disulfide isomers. The correct disulfide connectivity is very important for the biological activity6,7. However, the question that arises is whether there is more than one bioactive conformation of naturally occurring peptides and if so, which of those isomers possesses the highest biological activity? In the case of &#181;-conotoxins, the biological targets are voltage-gated sodium ion channels, and &#181;-PIIIA in particular is most potent for subtypes NaV1.2, NaV1.4 and NaV1.73.
The synthesis of disulfide-bridged peptides can be achieved using various methods. The most convenient method for the formation of disulfide bonds within a peptide is the so-called oxidative self-folding approach. Here, the linear precursor of the desired cyclic peptide is synthesized first using solid-phase peptide synthesis, which is after the cleavage from the polymeric support subjected to oxidation in a buffer system. Redox-active agents such as reduced and oxidized glutathione (GSH/GSSG) are often added to promote the formation of the disulfide bonds. The main disadvantage of buffer-supported self-folding is that the disulfide bonds are not formed selectively in a stepwise fashion. Compared to the native peptide, for which often only one specific disulfide isomer is described, it is possible to obtain numerous other isomers with this approach8. &#181;-PIIIA has already been shown to result in at least three differently folded isomers upon self-folding in a previous study3. The separation of such an isomer mixture is rather difficult due to similar retention times if using chromatographic purification methods9. The targeted synthesis of a specific isomer is therefore advantageous. To specifically produce an isomer with defined disulfide connectivity, a special strategy is required in which the disulfide bonds are successively closed. Therefore, the linear precursor carrying distinct protecting groups at the individual cysteine pairs is synthesized on the polymer support. After the elimination, the cysteine pairs are individually and successively deprotected and linked in an oxidation reaction to yield the desired disulfide bonds10,11,12,13,14,15,16. After the synthesis and the purification of the reaction product, it is required to confirm the identity and the disulfide connectivity by suitable analytical methods. Numerous analytical methods are available for the elucidation of the primary amino acid sequence, e.g., MS/MS, while the determination of the disulfide connectivity still remains much less investigated. Apart from the complexity of such multiple disulfide-bonded peptides, product-related impurities (e.g., from disulfide scrambling), due to sample preparation and work-up can further complicate analysis. In this paper, we show that the use of a combination of different analytical techniques is necessary to unequivocally clarify the identity of the disulfide bonds in the &#181;-PIIIA isomers. We have combined chromatographic methods with mass spectrometry and provided the same samples to NMR spectroscopy. In matrix-assisted laser desorption/ionization(MALDI) MS/MS analysis, we identified the disulfide bonds by using partial reduction and iodoacetamide derivatization because top-down analysis is not possible for this peptide. 2D NMR experiments were performed in order to obtain a three-dimensional structure of each isomer. Thus, by combining distinct sophisticated analytical methods, it is possible to elucidate properly the disulfide connectivity and three-dimensional structure of complex multiple disulfide-bonded peptides7.

<table border="0" cellpadding="0" cellspacing="0" style="width:979px;" width="979">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Fmoc Rink amide resin</td>
			<td style="width:185px;">Novabiochem</td>
			<td style="width:191px;">855001</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Pyr(Boc)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">A-3850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Arg(Pbf)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Asn(Trt)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Asp(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hyp(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FAA1627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Lys(Boc)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Ser(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gln(Trt)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Glu(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Trp(Boc)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Tyr(tBu)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">47623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Thr(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">His(Trt)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FDP1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorphosphat</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">8510060</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">DMF</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">D119</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">DCM</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td>D37</td>
			<td>Carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Piperidine</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td>A12442</td>
			<td>Flammable, Toxic, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">N-Methyl-Morpholin</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">224286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(Acm)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FAA1506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(Trt)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">E-2495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(tBu)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">trifluoruacetic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">74564</td>
			<td>Toxic, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">phenol</td>
			<td style="width:185px;">Merck</td>
			<td style="width:191px;">1002060</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">thioanisol</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:191px;">A14846</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ethanedithiol</td>
			<td style="width:185px;">Fluka Analytical</td>
			<td style="width:191px;">2390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">diethyl ether</td>
			<td style="width:185px;">VWR</td>
			<td style="width:191px;">100,921</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">tert-butanol</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:191px;">L12338</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">acetonitrile</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">A998</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">water</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">W5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">isopropanol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:191px;">ACRO42383</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium hydroxide</td>
			<td style="width:185px;">AppliChem</td>
			<td style="width:191px;">A6579,1000</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">iodoacetamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">I6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">iodine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">I0385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hydrochloric acid</td>
			<td style="width:185px;">Merck</td>
			<td style="width:191px;">110165</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ascorbic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">diphenylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">P35405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">anisol</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">96109</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">trichloromethylsilane</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">M85301</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sample dilution buffer</td>
			<td style="width:185px;">Laborservice Onken</td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium dihydrogen phosphate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">106370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">disodium hydrogen phosphate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">795410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(2-carboxyethyl)phosphine hydrochloride</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">C4706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">citric acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">251275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium citrate dihydrate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">W302600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris-acetate</td>
			<td style="width:185px;">Carl Roth,&#160;</td>
			<td style="width:191px;">7125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">E26282&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">peptide calibration standard II</td>
			<td style="width:185px;">Bruker Daltonics GmbH</td>
			<td style="width:191px;">8222570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;"></td>
			<td style="width:185px;"></td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Name of Equipment</td>
			<td style="width:185px;">Company</td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">solid-phase peptide synthesizer</td>
			<td>Intavis Bioanalytical Instruments AG</td>
			<td style="width:191px;">EPS 221</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:340px;">lyophilizer&#160;</td>
			<td>Martin Christ GmbH</td>
			<td style="width:191px;">&#160;Alpha 1-2 Ldplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">semipreparative HPLC</td>
			<td>Jasco</td>
			<td style="width:191px;">system PV-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Eurospher 100 C18 column (RP, 5 &#181;m particle size, 100 &#197; pore size, 250 x 32 mm)</td>
			<td style="width:185px;">Knauer</td>
			<td style="width:191px;">25QE181E2J</td>
			<td>purification of the linear peptide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:340px;">Vydac 218TP1022 column (RP C18, 10 &#181;m particle size, 300 &#197; pore size, 250 x 22 mm)</td>
			<td>Hichrom-VWR</td>
			<td style="width:191px;">HICH218TP1022</td>
			<td>purification of the oxidized peptide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:340px;">analytical HPLC&#160;</td>
			<td>Shimadzu</td>
			<td style="width:191px;">system LC-20AD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Vydac 218TP54 column (C18 RP, 5 &#181;m particle size, 300 &#197; pore size, 250 x 4.6 mm)&#160;</td>
			<td>Hichrom-VWR</td>
			<td style="width:191px;">HICH218TP54</td>
			<td>analytical column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ground steel target (MTP 384)</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">NC0910436</td>
			<td>MALDI preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">C18-concentration filter (ZipTip)</td>
			<td style="width:185px;">Merck KGaA</td>
			<td style="width:191px;">ZTC18S096</td>
			<td>MALDI preparation&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MALDI mass spectrometer</td>
			<td style="width:185px;">Bruker Daltonics GmbH</td>
			<td style="width:191px;">ultraflex III TOF/TOF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amino acid analyzer</td>
			<td>Eppendorf-Biotronik GmbH</td>
			<td style="width:191px;">LC 3000 system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NMR spectrometer Bruker Avance III</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">Bruker Avance III 600 MHz</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">computer program for molecular visualising</td>
			<td>YASARA Biosciences GmbH</td>
			<td style="width:191px;">Yasara structures</td>
			<td>NMR structure calculation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">computer program for MALDI data evaluation&#160;</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">flexAnalysis, BioTools</td>
			<td>MS/MS fragmentation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">analog vortex mixer</td>
			<td>VWR</td>
			<td style="width:191px;">VM 3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:191px;">5410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Centrifuge</td>
			<td>Hettich</td>
			<td style="width:191px;">EBA 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Rotational vacuum concentrator</td>
			<td style="width:185px;">Christ</td>
			<td style="width:191px;">2-18 Cdplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Analytical Balance</td>
			<td style="width:185px;">A&#38;D Instruments</td>
			<td style="width:191px;">GR-202-EC</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and structure determination of &#181;-conotoxin piiia isomers with different disulfide connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

15 different disulfide-bridged isomers of the &#181;-conotoxin PIIIA are synthesized and characterized in detail (Figure 1). Disulfide bonds are identified by partial reduction and subsequent MS/MS analysis (Figure 2). NMR analysis of the different isomers is carried out (Figure 3) to reveal the individual peptide structures. Notably, a combination of RP HPLC, MS/MS fragmentation, and NMR analysis is...

Watch this Scientific Journal Video about Synthesis and Structure Determination of Âµ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities at JoVE.com

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

This method can help answer key questions in the peptide synthesis field such as synthesizing disulfide rich peptides and determining their corresponding disulfide bridge pattern. The main advantage of this technique is that it allows for the selective synthesis of different disulfide bondend peptide isomers and their subsequent chemical and structural characterization. Generally individuals new to this method will struggle because each disulfide rich peptide behaves differently.Synthesis and analysis may have to be partially optimized for individual peptide or protein. Transfer the reagents listed in the text protocol to the corresponding vessels and place them in the appropriate rackets of the solid phase peptide synthesizer. Then, add 100 milligrams of dry resin to the reaction columns and put them in the racket of the synthesizer.Start the solid-phase peptide synthesis as detailed in the text protocol. After the synthesis is complete, lyophilized the resin overnight. Now cleave the peptide from the resin and perform deep protection of the side chains.To do so combine the dried resin in a 12 milliliter tube and cool it to zero degrees Celsius on ice. First, add 150 microliters of a scavenger mixture, then add one milliliter of 95%trifluoroacetic acid per 100 milligrams of resin. Leave the mixture gently shaking for three hours at room temperature.Now, filter the mixture through a glass frit. Collect the filtrate in tubes, individually filled with ice-cold diethyl ether at one milliliter of cleavage mixture per eight to 10 milliliters of diethyl ether. The peptide precipitates as a white solid.After rinsing and centrifuging the tubes as described in the text protocol, freeze-dry the peptides. To purify the linear precursor with semi preparative chromatography add approximately 70 milligrams of the crude peptide to a 15 milliliter tube. Then dissolve the crude peptide in 0.1%TFA and one-to-one Acetonitrile to water.Separate the peptide mixture using a gradient of zero to 50%Eluent B over 120 minutes at a flow rate of 10 milliliters per minute. Collect the fractions in individual tubes as they appear and prepare selected fractions for Mass Spectrometry and HPLC analysis as described in the text protocol. Freeze-dry the fractions and combine the peer fractions.Start the Selective formation of disulfide bonds by performing the first oxidation. To do so, dissolve 15 milligrams of the freeze-dried linear peptide in 105 milliliters of an isopropanol water mixture. Leave the mixture stirring on air under basic conditions for 12 to 48 hours.The stepwise oxidation procedure is very critical. Reaction controls are taken in all three oxidation steps to ensure individual disulfide rich formation. The third oxidation is the most crucial because disulfide scrambling can occur when the optimal reaction time is exceeded.Now perform the second oxidation. Dissolve 15 milligrams of the freeze-dried peptide in 105 milliliters of an isopropanol water, one molar hydrochloric acid mixture. Add 158 microliters of a 0.1 molar iodine solution in methanol to the solution.Stir the reaction at room temperature until the oxidation is completed. Then stop the reaction by adding 79 microliters of one molar ascorbic acid in water. Finally perform the third oxidation.Dissolve 15 milligrams of the freeze-dried peptide in 5.5 milliliters of TFA containing a scavenger mixture. Precipitate the peptide into tubes containing cold diethyl ether at one milliliter of reaction solution per eight to ten milliliters of diethyl ether. To perform peptide purification at 15 milligrams of the freeze-dried crude product to a 15 milliliter tube.Dissolve the crude product in the volume of the HPLC sample loop using a mixture of 0.1%TFA and one to one Acetonitrile to water. After vortexing until complete dissolution, centrifuge the solution from one minute at 3400 times G.Draw up the 3.6 milliliter mixture in a 5 milliliter syringe and inject the sample without any air bubbles into the injection loop. Start the injection into the HPLC system.Purify the peptide mixture using a gradient of zero to 50%Eluent B over 120 minutes at a flow rate of 10 milliliters per minute. Collect the fractions in individual tubes as they appear. After the run is complete, prepare selected fractions for MS and HPLC analysis as described in the text.Freeze-dry all fractions and store them at minus 20 degrees Celsius. To perform analytical HPLC, transfer samples of the peptide fractions or reaction controls into an HPLC vial and dissolve it in the mixture of 0.1%TFA in one to one Acetonitrile to water. Place the HPLC vial into the auto-sampler of the analytical reverse phase HPLC set to inject 250 microliters of each sample.Use a gradient elution system of 0.1%TFA in water as Eluent A in 0.1%TFA in Acetonitrile as a Eluent B.Analyze the peptide using a gradient of 10%to 40%Eluent B over 30 minutes at a flow rate of 1.0 milliliter per minute. To perform amino acid analysis, transfer 100 micrograms of the pure peptide to a 1.5 milliliter micro-centrifuge tube and dissolve the powder in 200 microliters of six molar HCL. After transferring the solution to a glass ampule, close the ampule by heating the neck with a bunsen burner flame.Then place a glass tube holding the ampule in a heating block for 24 hours at 110 degrees Celsius for hydrolysis. After 24 hours, open the ampule and transfer the solution into a two milliliter micro-centrifuge tube. Centrifuge the solution for six hours at sixty degrees Celsius and 210 times G in a rotational vacuum concentrator.Then dissolve the hydrolyzed product in 192 microliters of the sampled dilution buffer and transfer the solution into a micro centrifugal filter. After centrifuging the sample for one minute at 2300 times G, transfer 100 microliters of the filtrate into an amino acid analysis sample tube. Place the tube into the amino acid analyzer and start the analysis.During this past reduction protocol, it is crucial to take several reaction controls in order to obtain twice or four times kappa mido methylated species which are indispensable for subsequent disulfide analysis via mass spectrometry. First dissolve 600 micrograms of the pure peptide in 1.2 milliliters of 0.05 molar citrate buffer containing TCEP. Incubate the mixture at room temperature taking several 100 microliter reaction control samples ranging from zero to 30 minutes.Mix the samples in a 1.5 milliliter micro-centrifuge tube with 300 microliters of alkylation buffer to stop the reaction and perform carbonyl methylation of the free thiol groups. Stop the reaction after five minutes by adding 100 microliters of 10%TFA and water and store the samples on dry ice. Now perform HPLC on the samples as detailed in the text protocol.Transfer a small amount of each collected fraction to a 1.5 milliliter micro-centrifuge tube for MS/MS analysis of the oxidized forms. Dissolve the remaining peptide in 0.1%TFA in water and add an appropriate volume over a 100 millimolar TCEP solution to get a final TCEP concentration of 10 millimolar. At this point, selective cysteine alkylation can be checked via peptide sequencing.With the two peptides in the middle, a determination of the disulfide bridge pattern is possible. NMR analysis of the different isomers is carried out to reveal the individual peptide structures. The 20 structures with the lowest energies as well as the disulfide connectivity of the different isomers are shown.Notably, a combination of reversed-phase HPLC MS/MS fragmentation and NMR analysis is required for unambiguous identification of the disulfide connectivity. The comparison of the root mean square deviation value clarified that a rigid peptide mostly leads to a better resolved NMR structure. This video should give you an example how to selectively synthesize peptides with a desired disulfide bond pattern.It also shows the possibility to check the correct disulfide connectivity via tender mass spectrometry and to obtain a three-dimensional structure via NMR spectroscopy. After its development, this technique paved the way for researchers in the field of peptide synthesis to explore the importance of disulfide bond patterns for the three-dimensional structure and consequently the activity of such peptide isomers. Following this procedure, other methods like activity ASAS can be performed in order to get insight into structure activity relationships of different peptide isomers at the specific biological target.Though this method can provide insight into the synthesis and analysis of conotoxins, it can also be applied to other disulfide bridge peptides and proteins such as the fenzens, disulfide-rich animal toxins and other multiple cysteine containing molecules.

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12.4K Görüntüleme.  University of Bonn. Bu yöntem, disülfür zengin peptidlerin sentezlenmesi ve buna karşılık gelen disülfür köprü deseninin belirlenmesi gibi peptid sentezi alanındaki temel soruların yanıtlandırılmasına yardımcı olabilir. Bu tekniğin en büyük avantajı, farklı disülfür bondend peptid izomerlerinin seçici sentezine ve sonraki kimyasal ve yapısal karakterizasyonuna olanak sağlamasıdır. Her disülfür zengin peptid farklı davrandığını, çünkü genellikle bireylerin bu yönteme yeni ...