Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Summary

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.

Abstract

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 µ-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.

Introduction

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 studies^2,3,4. Apart from the primary amino acid sequence that influences the overall conformation, disulfide bonds significantly stabilize the structure of cysteine-rich peptides⁵. Multiple disulfide-bridg....

Protocol

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 ZRLCCGFOKSCRSRQCKOHRCC-NH₂ using a standard protocol for 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. Apply the following protected amino acids: Pyr(Boc ....

Results

15 different disulfide-bridged isomers of the µ-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.......

Discussion

The method described herein for the synthesis of cysteine-rich peptides such as µ-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.......

Materials

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