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Solid Phase Peptide Synthesis

AAPPTec's Guide to Solid Phase Peptide Synthesis is an introduction to solid phase peptide synthesis It discusses how solid phase peptide synthesis is performed, the amino acid derivatives, resin and reagents used in peptide synthesis, and some of the common problems in solid phase peptide synthesis and how to avoid them, plus also suggested procedures and guides to choosing resins and planning a successful peptide synthesis.

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I. Introduction - Publications

II. Brief Outline and History of Solid Phase Peptide Synthesis

A. History

B. Overview of Solid Phase Peptide Synthesis

1. General Solid Phase Peptide Synthesis Scheme

2. Selective Deprotection

a. Boc/Bzl Protection

b. Boc Deprotection Mechanism

c. Fmoc/tBu Protection

d. Fmoc Deprotection Mechanism

e. Ligation and Fragment Condensation

III. Equipment for Solid Phase Peptide Synthesis

A. Manual Synthesis

B. Automated Synthesizers

IV. Resins for Solid Phase Synthesis

A. Core Resins

1. Polystyrene
2. Merrifield Resin
3. Hydroxymethyl Resin
4. Amino Core Resins

B. Linked Resins

1. Linkers
2. PAM Resin
3. Wang Resin
4. Amide/Amine Forming Resins for Fmoc/tBu
5. MBHA Resin
6. Trityl and 2-Cl-Trityl Resin
7. Nucleophile Labile Resins
8. DHP Resins
9. Weinreb Resin
10. Substrate-Attached Resins

V. Solvents for Solid Phase Peptide Synthesis

VI. Amino Acid Derivatives

A. Arginine

B. Aspartic Acid and Glutamic Acid

C. Asparagine and Glutamine

D. Cysteine and Penicillamine

E. Histidine

F. Lysine, Ornithine, 2,4-Diaminobutanoic Acid and 2,3-Diaminopropanoic Acid

G. Methionine

H. Serine, Threonine, and 4-Hydroxyproline

I. Tryptophan

J. Tyrosine

VII. Coupling Reagents

A. Carbodiimides





F. Other Coupling Reagents

VIII. Monitoring of Coupling and Capping

IX. Aggregation, Difficult Sequences, and Side Reactions

A. Aggregation

B. Racemization

C. Side Reactions

1. Diketopiperidine Formation
2. Aspartamide Formation
3. Pyroglutamate Formation
4. 3-(1-Piperidinyl)alanine Formation
5. Guanidinylation
6. Transfer of Sulfonyl Protecting Groups
7. Oxidation of Methionine
8. N->O Shift

D. Side Reactions During HF Cleavage

1. Homoserine Lactone Formation
2. Glutamic Acid Side Reactions
3. Asp-Pro Cleavage

X. Peptide Modification

A. Cyclization

B. Stapled Peptides

C. N-Methylation of Peptide Backbone

D. Phosphorylation

E. Myristylation/Palmitylation

F. Glycosylation

G. Isoprenylation

H. PEGylation

I. Biotinylation

J. Fluorescent Labeling

K. Caged Peptides

L. MAP Peptides

M. Thioesters

XI. Planning a Peptide Synthesis

A. N- and C-Terminal Functionality

B. Boc vs Fmoc

C. Selecting a Resin for Peptide Synthesis

D. Selecting Amino Acid Derivatives

E. Planning the Synthesis of Peptides Containing Multiple Disulphide Bonds

F. Storing and Using Amino Acid Solutions

XII. Technical Notes

A. Attaching the First Residue to Resin

1. Merrifield Resin
2. Activation of Trityl Resins
3. Attachment of Amino Acids to Trityl Resins
4. Attachment of Carboxylic Acids to Hydroxy-Substituted Resins
5. Attachment of Amino Acids to Fmoc-Protected Amide Forming Resins
6. Attachment of Boc-Amino Acids to BHA or MBHA Resins
7. Alcohol Coupling to Carboxypolystyrene

B. Measuring Substitution of Fmoc-Amino Acid Resins

C. Standard Coupling Procedures

1. N-Terminal Deprotection

a. Boc Deprotection

b. Fmoc Deprotection

c. Fmoc Deprotection with DBU

2. Converting CHA and DCHA Salts to Free Acids

3. Coupling

a. DIC/HOBt Coupling

b. EDC Coupling

c. BOP Coupling

d. PyBOP Coupling


f. Fast Boc Deprotection with In Situ Coupling

g. N-Methyl Amino Acid Coupling with PyBrOP

h. TSTU Coupling in Aqueous Solvents

i. Fragment Coupling with TBTDU

j. Solid Phase Coupling with DEPBT

k. Solution Phase Coupling with DEPBT

l. Head to Tail Cyclization with DEPBT

4. Coupling Tests

a. Kaiser Test (Ninhydrin Test)

b. Isatin Test

c. Chloranil Test

5. Capping Procedure

D. Cleavage

1. Cleavage from Merrifield Resin

a. Standard HF Cleavage

b. Low-High HF Cleavage

c. Standard Trifluoromethanesulfonic Acid Cleavage

d. Low-High Trifluoromethanesulfonic Acid Cleavage

e. TMSOTf Cleavage

2. Wang Resin Cleavage

a. TFA Cocktail Procedure

b. TMSBr Procedure

3. HMPA Resin Cleavage

4. Rink Amide Resin Cleavage

5. Cleavage from Sieber Amide Resin

6. Cleavage of Protected Peptides from 2-Clorotrityl Resin

7. Cleavage from Oxime Resin

a. Cleavage to Peptide Acid

b. Cleavage to Peptide Ester

c. Cleavage to Peptide Amide

8. HMBA-MBHA Resin Cleavage

a. Cleavage to Peptide Acid

b. Cleavage to Peptide Ester

c. Cleavage to Peptide Amide

9. Weinreb Resin Cleavage to Aldehydes

9. Alcohol Cleavage from DHP Resin

11. Alcohol Cleavage from Carboxypolystyrene

a. Sodium Carbonate Cleavage

b. Sodium Methoxide Cleavage

E. Cleavage Cocktails

1. “Odorless” Reagent B

2. Reagent H for Methionine Containing Peptides

3. Reagent K for Peptides Containing Cys, Trp, Met, or Tyr

4. Low Odor Reagent L

5. Reagent R for Arginine Containing Peptides

F. Side-chain Deprotection

1. Arg(Mtr)

2. Arg(Pbf) and Arg(Pmc)

3. Cys(Acm)

4. Cys(tBu)

5. Cys(Trt)

6. His(DNP)

7. Allyl and Aloc

8. Dde and ivDde

9. Trp(CHO)

10. Met(O)

XIII. Post Cleavage Purification and Analysis

A. Precipitation and Isolation

B. Yield Calculation

C. HPLC Analysis

D. HPLC Purification

E. Removal of TFA

XIV. Storage and Handling of Peptides

XV. Dissolving Peptides


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