Fmoc Solid Phase Peptide Synthesis A Practical Approach

by Chan, W. C.; White, Peter D.

• ISBN13: 9780199637249
• ISBN10: 0199637245
• Format: Paperback
• Copyright: 2000-03-02
• Publisher: Oxford University Press

Summary

In the years since the publication of Atherton and Sheppard's volume, the technique of Fmoc solid-phase peptide synthesis has matured considerably and is now the standard approach for the routine production of peptides. The basic problems outstanding at the time of publication of this earlier work have now been, for the most part, solved. As a result, innovators in the field have focussed their efforts to develop methodologies and chemistry for the synthesis of more complex structures. The focus of this new volume is much broader, and covers not only the essential procedures for the production of linear peptides but also more advanced techniques for preparing cyclic, side-chain modified, phospho- and glycopeptides. Many other methods also deserving attention have been included: convergent peptide synthesis; peptide-protein conjugation; chemoselective ligation; and chemoselective purification. The difficult preparation of cysteine and methionine-containing peptides is also covered, as well as methods for overcoming aggregation during peptide chain assembly and a survey of available automated instrumentation.

Table of Contents

List of Contributors	xvii
Abbreviations	xxi
Introduction---a retrospective viewpoint	1	(8)
R. C. Sheppard
Basic principles	9	(32)
Peter D. White
Weng C. Chan
The solid phase principle	9	(2)
Merrifield SPPS	11	(1)
Fmoc/tBu SPPS	11	(30)
Resins	13	(2)
Linkers	15	(11)
Side-chain protecting groups	26	(1)
First residue attachment	26	(1)
Coupling step	27	(1)
N-Fmoc deprotection reaction	27	(3)
Aggregation	30	(1)
Enantiomerization	31	(1)
Side reactions	32	(4)
Cleavage reaction	36	(1)
Limitations	36	(1)
References	37	(4)
Basic procedures	41	(36)
Weng C. Chan
Peter D. White
Introduction	41	(1)
Manual synthesis	41	(1)
Resin handling	41	(3)
Resin functionalization	44	(1)
Attachment of the first residue	44	(7)
Hydroxymethyl-based resins	44	(6)
Trityl-based linkers	50	(1)
Aminomethyl-based linkers	51	(1)
Fmoc removal	51	(1)
Coupling methods	52	(8)
DIC/HOBt	53	(1)
Active esters	54	(1)
Symmetrical anhydride	55	(2)
Aminium/phosphonium activation methods	57	(2)
Acid fluorides	59	(1)
Assembly of the peptide chain	60	(1)
Analytical procedures	61	(3)
Resin tests	61	(1)
Bromophenol blue monitoring	62	(1)
Fmoc determination	62	(1)
HPLC analysis	63	(1)
TFA-mediated cleavage	64	(13)
Preparing the resin for cleavage	64	(1)
Cleavage reactions releasing fully deprotected peptides	65	(5)
Peptide isolation	70	(2)
Monitoring the cleavage reaction	72	(1)
Release of fully protected peptides from hyper-acid labile supports with 1% TFA	73	(1)
References	74	(3)
Preparation and handling of peptides containing methionine and cysteine	77	(38)
Fernando Albericio
Ioana Annis
Mirian Royo
George Barany
Introduction	77	(1)
Methionine	78	(3)
Cysteine	81	(34)
Cysteine protection	81	(6)
Solid phase synthesis of cysteine-containing peptides	87	(4)
Formation of disulphides	91	(18)
References	109	(6)
Difficult peptides	115	(22)
Martin Quibell
Tony Johnson
Introduction	115	(1)
Difficult peptides--an overview	115	(4)
Background	115	(2)
Identifying the effects of aggregation	117	(1)
Effect of resins, solvents, and additives on aggregation	118	(1)
Predicting difficult peptide sequences	119	(3)
The N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone amide protecting group	122	(15)
Development and preparation of (Hmb) amino acid derivatives	122	(3)
Incorporation of N-(2-hydroxy-4-methoxybenzyl)amino acid residues	125	(2)
Site selection for Hmb back bone protection	127	(1)
Improved difficult peptide syntheses through Hmb backbone protection	128	(3)
Use of Hmb protection to increase solution solubility	131	(1)
References	132	(5)
Synthesis of modified peptides	137	(46)
Sarah L. Mellor
Donald A. Wellings
Jean-Alain Fehrentz
Marielle Paris
Jean Martinez
Nicholas J. Ede
Andrew M. Bray
David J. Evans
G. B. Bloomberg
C-terminal modifications	137	(29)
Peptidyl N-alkyl amides	137	(4)
Use of the 4-hydroxymethylbenzoic acid linkage agent for the synthesis of C-terminal modified peptides	141	(8)
Peptide hydroxamic acids	149	(4)
Peptide aldehydes by solid phase synthesis	153	(8)
Synthesis of C-terminal peptide aldehydes on the Multipin™ system using the oxazolidine linker	161	(5)
N-terminal modifications	166	(3)
Biotinylation	167	(1)
Reductive alkylation	168	(1)
Side-chain modifications	169	(14)
Introduction	169	(1)
Strategy design in the synthesis of atypical peptides	169	(9)
References	178	(5)
Phosphopeptide synthesis	183	(12)
Peter D. White
Introduction	183	(1)
Building block approach	183	(4)
Introduction	183	(1)
Practical considerations	184	(1)
Illustrative examples	185	(2)
Global phosphorylation	187	(5)
Introduction	187	(2)
General protocol for post-synthetic phosphorylation	189	(1)
Illustrative examples	190	(1)
Thiophosphorylated peptides	191	(1)
Analysis of phosphopetides	192	(3)
HPLC analysis	192	(1)
Mass spectroscopy	192	(1)
References	193	(2)
Glycopeptide synthesis	195	(20)
Jan Kihlberg
Introduction	195	(2)
Strategic considerations in glycopeptide synthesis	197	(1)
Formation of glycosidic linkages to amino acids	198	(2)
Choosing protective groups for glycosylated amino acids	200	(3)
Protection of the α-amino group	200	(1)
Protection of the α-carboxyl group	201	(1)
Protection of the carbohydrate hydroxyl groups	201	(1)
Suitable linkers and resins	202	(1)
Preparation of glycosylated amino acids	203	(6)
Synthesis of a glycopetide from HIV gp120	209	(6)
References	211	(4)
Convergent peptide synthesis	215	(14)
Kleomenis Barlos
Dimitrios Gatos
Introduction	215	(1)
Strategy in convergent synthesis	215	(1)
Solid phase synthesis of protected peptide fragments	216	(4)
Fragment selection	216	(1)
Synthesis of protected peptide fragments	216	(4)
Solid phase fragment condensation	220	(3)
Esterification of the C-terminal fragment on 2-chlorotrityl chloride resin	220	(1)
Activation and condensation of protected peptide fragments	221	(2)
Phase and direction change	223	(4)
Fragment condensation in solution	223	(2)
Two-directional synthesis. Attachment of fragments on resins of the trityl-type through an amino acid side chain functional group	225	(2)
Deprotection, purification, and purity determination of the synthetic peptides	227	(2)
References	227	(2)
Methods of preparing peptide---carrier conjugates	229	(14)
Jan W. Drijfhout
Peter Hoogerhout
Introduction	229	(1)
Homobifunctional cross-linking	229	(2)
Heterobifunctional cross-linking	231	(5)
Conjugation of thiol-containing peptides to proteins	231	(3)
Conjugation of peptides to thiolated carriers	234	(2)
MAP-core constructs as peptide-carriers	236	(7)
Sequential synthesis of MAPs	237	(1)
Synthesis of MAPs by fragment condensation	237	(2)
References	239	(4)
Chemoselective and orthogonal ligation techniques	243	(22)
James P. Tam
Y.-A. Lu
Introduction	243	(1)
Chemoselective non-amide ligation	244	(8)
Thiol chemistry	245	(2)
Carbonyl chemistry	247	(5)
Orthogonal amide ligation	252	(13)
Carbonyl chemistry	252	(6)
Thiol chemistry	258	(4)
References	262	(3)
Purification of large peptides using chemoselective tags	265	(12)
Paolo Mascagni
Introduction	265	(1)
Purification of large polypeptides using Fmoc-based chromatographic probes	266	(2)
The concept of selective and reversible labelling	266	(2)
Capping of unreacted polypeptide chains in SPPS	268	(1)
RP-HPLC using lipophilic chromatographic probes	269	(5)
Affinity chromatography using biotinylated chromatographic probes	274	(3)
References	276	(1)
Instrumentation for automated solid phase peptide synthesis	277	(26)
Linda E. Cammish
Steven A. Kates
Introduction	277	(2)
Batchwise peptide synthesis	279	(8)
PE Biosystems Model 433A peptide synthesis system	280	(3)
Protein Technologies, Inc., SONATA/Pilot™ peptide synthesizer	283	(1)
Protein Technologies Inc., Model PS3™ peptide synthesizer	283	(1)
Advanced ChemTech Model 90 peptide synthesizer	284	(2)
Advanced ChemTech Model 400 production-scale synthesizer	286	(1)
ABIMED EPS 221 synthesizer	287	(1)
Continuous-flow peptide synthesis	287	(4)
PE Biosystems Pioneer™ peptide synthesis system	289	(2)
Multiple peptide synthesis systems	291	(8)
PE Biosystems Pioneer MPS option	291	(1)
Protein Technologies, Inc., SYMPHONY/Multiplex™ peptide synthesizer	292	(1)
Advanced ChemTech Models 348, 396, and 357 bimolecular synthesizers	293	(1)
ABIMED AMS 422 multiple peptide synthesizer	294	(2)
ABIMED ASP 222 Auto-Spot Robot	296	(1)
ZINSSER ANALYTIC SMPS 350 multiple peptide synthesizer	297	(1)
ZINSSER ANALYTIC SOPHAS solid phase synthesizer	298	(1)
SHIMADZU PSSM-8 peptide synthesizer	298	(1)
Conclusions	299	(4)
References	300	(3)
Manual multiple synthesis methods	303	(26)
B. Dorner
J. M. Ostresh
R. A. Houghten
Ronald Frank
Andrea Tiepold
John E. Fox
Andrew M. Bray
Nicholas J. Ede
Ian W. James
Geoffrey Wickham
Simultaneous multiple peptide synthesis---the T-bag method	303	(2)
Introduction	303	(1)
The T-bag method	303	(2)
Multiple peptide synthesis with the SPOT-technique	305	(9)
Introduction	305	(3)
Synthesis of peptide SPOT-arrays	308	(5)
Applications of peptide SPOT-arrays	313	(1)
Manual multipeptide synthesis in block arrays	314	(5)
Hardware	314	(1)
Chemistry	315	(2)
Software	317	(2)
Synthesis of peptides by the Multipin™ method	319	(10)
Introduction	319	(1)
Linkers	320	(1)
Peptide synthesis	321	(4)
References	325	(4)
Appendices	329	(12)
A1 Equipment and reagents for peptide synthesis	329	(2)
A2 List of suppliers	331	(6)
A3 Useful tables	337	(4)
Index	341