Automated solid-phase peptide

GE Reid, RJ Simpson. Automated solid-phase peptide synthesis use of 2-(17f-ben-zotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids. Anal Biochem 200, 301, 1992. 

W van den Nest, S Yuval, F Albericio. Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis. J Pept Sci 7, 115, 2001. 

Caged peptides (NPY and angiotensin II) containing Tyr[Bzl(2-N02) were synthesized on an automated solid-phase peptide synthesizer using Fmoc chemistry and cleaved from the resin by treatment with TFA. Crude peptides were purified by semipreparative HPLC with a gradient phase of 0.1% TFA and MeCN. For fractions containing peptides, the eluant was not passed through the UV monitor to avoid potential photolysis. The collected peptide was examined by analytical HPLC and MS. 

Less reactive than acyl halides, but still suitable for difficult couplings, are symmetric or mixed anhydrides (e.g. with pivalic or 2,6-dichlorobenzoic acid) and HOAt-derived active esters. HOBt esters smoothly acylate primary or secondary aliphatic amines, including amino acid esters or amides, without concomitant esterification of alcohols or phenols [34], HOBt esters are the most commonly used type of activated esters in automated solid-phase peptide synthesis. For reasons not yet fully understood, acylations with HOBt esters or halophenyl esters can be effectively catalyzed by HOBt and HOAt [3], and mixtures of BOP (in situ formation of HOBt esters) and HOBt are among the most efficient coupling agents for solid-phase peptide synthesis [2]. In acylations with activated amino acid derivatives, the addition of HOBt or HOAt also retards racemization [4,12,35]. 

Amino acids were added to the N -Boc-D-Aia-0-Resin (1.0 mmol of resin was replaced in the reaction vessel of 5.0 L Vega 296 automated solid phase peptide synthesizer in the following sequence ... 

Fig. 15 The newly introduced principle of single amino acid chelators (42, SAAC) which can directly be subjected to automated solid phase peptide syntheses...

While chemical synthesis is mostly an art, with specialized reactions for both inorganic and inorganic synthesis, the complexities of biochemistry have nurtured specialized instruments that can split or assemble biomolecules. An automated solid-phase peptide synthesizer was introduced by Merrifield15 in 1963 this allows for the facile synthesis of oligopeptides (up to 100 amino acid units) [2], The enzyme DNA polymerase I was discovered by Komberg16 in 1957 this allowed the assembly of DNA from fragments [3]. 

A peptide having the identical sequence was S3mthesized using standard automated solid phase peptide techniques (18). 

Assay of Truncated Peptides. The truncated forms of allatostatin 4 were synthesized on a Pharmacia-LKB Biolynx model 4170 automated solid-phase peptide synthesizer by The Biotechnology Service Centre, Department of Clinical Biochemistry, Banting and Best Institute, Toronto, Canada. The assays comparing the activity of allatostatin 4 with the two analogues were carried out as for the HPLC eluates. 

Green fluorescent protein as a marker Section 4.3.5 Immunoelectron microscopy Section 4.3.5 Automated solid-phase peptide synthesis Section 4.4 Nuclear magnetic resonance spectroscopy Section 4.5.1... 

R. Bruce Merrifield was born in 1921 and received a B.S. and a Ph.D. from the University of California, Los Angeles. He is a professor of chemistry at Rockefeller University. Merrifield received the 1984 Nobel Prize in chemistry for developing automated solid-phase peptide synthesis. 

In the Merrifield method, the C-terminal amino acid is covalently attached to a solid support contained in a column. Each N-terminal blocked amino acid is added one at a time, along with other needed reagents, so the protein is synthesized from the C-terminal end to the N-terminal end. Notice that this is opposite to the way proteins are synthesized in nature (from the N-terminal end to the C-terminal end Section 27.13). Because it uses a solid support and is automated, Merrifield s method of protein synthesis is called automated solid-phase peptide synthesis. 

L-amino acid (p. 964) amino acid analyzer (p. 970) amino acid residue (p. 959) anion-exchange resin (p. 970) antiparallel )S-pleated sheet (p. 990) automated solid-phase peptide synthesis (p. 980)... 

First we shall consider reactions for traditional chemical synthesis of peptides and then we look at reactions used in automated solid-phase peptide synthesis. The method for solid-phase peptide synthesis was invented by R. B. Merrifield (Rockefeller University), for which he earned the 1984 Nobel Prize in Chemistry. Solid-phase p>eptide synthesis reactions are so reliable that they have been incorporated into machines called peptide synthesizers (Section 24.7D). 

ROHR" FIGURE 24.8 A method for automated solid-phase peptide synthesis. 

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