polymerization
Peptide-resin aggregation can lead to slow or incomplete deprotection and incomplete coupling. A sign of aggregation is the inability of the peptide resin to swell. It is attributed to the self-association of peptides through hydrogen bonds. Aggregation cannot be reliably predicted from sequence data, although hydrophobic sequences are more likely to aggregate, and aggregation less likely before the fifth or sixth residue or after the twenty-first residue.
If aggregation becomes problematic, there are several different steps that can be taken to break the hydrogen bonds that cause aggregation. Some actions that can be taken include:
- If Fmoc deprotection is slow or incomplete, switch to DBU in the deprotection reagent. (DBU agreement)
- If using Boc/Bzl protection, switch to a neutralization-in-place protocol.
- Change to N-methylpyrrole (NMP) or add dimethyl sulfoxide (DMSO) to the solvent.
- Sonicate the reaction mixture.
- coupled with higher temperatures.
- Add chaotropic salts like CuLi, NaClO4or KSCN.
- Add nonionic detergent or ethylene carbonate (the magic mix).
- to usemicrowave irradiation.
If these measures do not significantly improve coupling, it may be advantageous to resynthesize the peptide on a low substituted or other resin such as TentaGel or SURE™.
The introduction of structural elements that disrupt the hydrogen bonds of the peptide backbone can effectively overcome the synthetic problems associated with difficult or long peptide sequences.pseudoprolina,i peptideySpine Protection Groupare elements used alone or in combination to reduce aggregation during solid phase synthesis. By reducing aggregation, these structures also increase the solubility of the cleaved peptides, facilitating purification of crude synthetic peptides. After the desired peptide has been purified, simple processing will provide the native peptide.
Esterification of serine or threonine residues effectively terminates the aggregation. If the peptide contains any of these residues, the corresponding depsipeptide can be prepared and weakly base rearranged into the desired peptide. The protocol was recently adapted to allow fully automated synthesis of long-chain depsipeptides, including the esterification procedure.
to useSpine Protection GroupFor example, a 2-hydroxy-4-methoxybenzyl (Hmb) or 2,4-dimethoxybenzyl (Dmb) group on the alpha nitrogen of an amino acid residue will prevent hydrogen bonding. Incorporation of a Hmb residue every 6 to 7 residues will effectively disrupt aggregation. Protection of the Hmb or Dmb backbone also prevents the formation of asparagine and the by-products produced.
Since prolines in peptide sequences are known to disrupt aggregation, the use ofpseudoprolinaThreonine-serine shunt is another strategy to disrupt aggregation. Pseudoprolines have been shown to be very effective in disrupting aggregation and allowing gradual synthesis of long peptides. Treatment with TFA, which cleaves the peptide from the resin, also converts the pseudoprolines to the corresponding serine or threonine residues.
careers
Activation of the protected amino acids results in some degree of racemization. Epimerization occurs through the mechanism shown below (Figure 1). addhobbit,6-Cl-HOBtohedInhibits racemization. Histidine and cysteine are particularly susceptible to racemization. Protection of the pi-imidazole nitrogen of the histidine side chain with a methoxybenzyl group greatly reduces racemization. Various reduced racemization protocols for the coupling of cysteine residues have been evaluated and compared.
Copper(II) chloride withhobbitIt has been used in solution phase peptide coupling to inhibit racemization. recently CuCl2It was reported to be efficient in solid phase synthesis using the unusual amino acid 4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine.
Figure 1 - Racemization mechanism
side effects
dicetopiperidindannelse
This side reaction occurs at the dipeptide stage and is more likely to occur in Fmoc-based synthesis. The formation of diketopiperazines is particularly common when proline is one of the first two residues.
Figure 2 – Formation of diketopiperazine
In Boc-based syntheses, the formation of diketopiperazines can be suppressed by using an in situ neutralization protocol. If you use the Fmoc/tBu protection strategy, i2-clortritilcloridharpiksIt is preferred when proline, pipecolic acid or TIC is one of the first two amino acids. The steric majority of the 2-chlorotrityl moiety inhibits the formation of diketopiperazines.
Another option is to add the second and third amino acid residues as dipeptide units, thus avoiding the dipeptide-resin intermediate. This strategy is limited by the availability of suitable dipeptides. A third option is to couple an N-trityl protected amino acid at the second position. The trityl groups are then removed with dilute TFA, yielding a protonated dipeptide resin, which can then be coupled via an in situ neutralization protocol.
Aspartame formation
Aspartime formation is particularly common in peptides containing Asp-Gly, Asp-Ala or Asp-Ser sequences. This side reaction can occur under acidic or basic conditions. Aspartame can regenerate a mixture of α- and β-linked peptides (Figure 7). In an Fmoc-based synthesis, piperidine can open azarthimide to piperidine. addhobbitarrivepiperidineThe deprotection solution will reduce the formation of asparagine. A specific cleavage protocol has been developed that reduces the formation of aspartimide. In Boc synthesis, useβ-cyclohexyl esterratherβ-bencilesterAspartic acid significantly reduces the amount of asparaginimine formed.
Asparagine formation can be blocked by introducing a protecting group at the α-nitrogen of the amino acid before aspartic acid in peptide synthesis. The groups 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4-dimethoxybenzyl (Dmb) have been used for this purpose in Fmoc chemistry. These groups prevent asparagine formation during synthesis and are removed when the peptide is cleaved from the resin by TFA treatment. Linkage to Dmb-protected amino acids can be difficult. SomeFmoc-AA-(Dmb)Gly-OH-dipéptidocommercially available.
This herepseudoprolinaFormation from serine is also effective in blocking asparagine imine formation in the Asp-Ser sequence, as shown in a semisynthetic preparation of the E. coli ribonucleotide reductase R2 subunit. Danishefsky has shown that pseudoproline and aspartic acid residues at n+2 also prevent asparagine formation.[30]
Figure 3 – Formation of aspartame
pyroglutamate formation
The N-terminal glutamine residue can undergo base-catalyzed cyclization to form pyroglutamic acid. As with the aspartic acid derivatives, addhobbitThe deprotection solution suppresses this side reaction.
3-(1-piperidinyl)alanine formation
This by-product is formed when peptides containing a C-terminal cysteine are prepared by the Fmoc/tBu protocol. Base-catalyzed removal of the protected thiol produces a dehydroalanine residue, which in turn adds piperidine. This byproduct can be confirmed by mass spectrometry as a mass change of +51. Use of space and large volumetrityl protecting groupThis by-product will be minimized but not eliminated.
Figure 4 – Formation of 3-(1-piperidinyl)alanine
Guanidinylering
The urea/ammonium coupling reagent reacts with the deprotected N-terminus of the peptide resin to form a guanidine residue that irreversibly terminates the peptide chain. Guanylation can be avoided by preactivating the protected amino acids with stoichiometric amounts of coupling reagents before adding the solution to the peptide resin. In situ neutralization inhibits guanidination in a protocol based on Boc protection.
Figura 5 - Guanidinylering
Transfer of the sulfonyl protecting group from Arg to Trp
During the final cleavage/deprotection of the peptide resin, the sulfonyl protecting group can be transferred from an arginine residue to a tryptophan residue. The amount of byproducts formed depends on the protecting group and the distance between the tryptophan and arginine residues. The use of a cleavage mixture containing a cleaning agent can reduce the amount of by-products formed.Reagent KyReactive Rare some examples of cleavage mixtures that are used when arginine residues are present.
The most efficient way to prevent migration of the sulfonyl protecting group to tryptophan during cleavage is to use an indole-protected tryptophan derivative:Boc-Trp(Para)-OHBoc/Bzl-based synthesis andFmoc-Trp(Boc)-OHIn synthesis based on Fmoc/tBu.
methionine oxidation
The thioether on the methionine side chain is readily oxidized to sulfoxide under acidic conditions. Addition of dithiothreitol (DTT) to the lysis mixture will inhibit oxidation. Alternatively, the oxidized peptides can be reduced to the desired peptide after cleavage. In some cases, methionine sulfoxide was used instead of methionine for peptide synthesis. This strategy is used when methionine residues are particularly susceptible to oxidation. Crude peptides are purified in the oxidized form and then reduced to their native form after purification. This leads to easier purification and higher recovery of the desired peptide.
change NO
Peptides containing serine or threonine residues can undergo acid-catalyzed acyl-N-O transfer. Treatment with a base such as ammonia reverses the reaction.
Bireaction under lysis
Formation of homoserine lactone during HF cleavage
The tert-butyl cation formed by deprotection of a tert-butyl-based protecting group can alkylate the thioether side chain of the C-terminal methionine, followed by cyclization to produce a homoserine lactone. This side reaction can be prevented by removing all tBu-based protecting groups prior to HF cleavage.
Figure 6 – Formation of homoserine lactone
glutamate side effects
Deprotection of glutamic acid residues during HF cleavage results in protonation of HF to form an acyl ion and dehydration of the deprotected carboxyl residue. The acyl ion can cyclize to produce a pyroglutamine residue, or it can react with scavengers such as anisole to form aryl ketones.
Figure 7 – Glutamate side reactions during HF cleavage
Asp-Pro split
Cleavage of this bond has been reported during cleavage by HF.
Cys side reactions during cleavage of Wang's resin
Cysteine residues with acid-labile protecting groups, such as trityl (Trt), 4-methoxytrityl (Mmt), or the recently reported tetrahydropyranyl (Thp), can form S-alkylated by-products as a result of cleavage of the linkers of Rink Amide or Wang resin. The 4-hydroxybenzylated byproduct arising from cleavage of the Wang linker is particularly common when the Cys residue is at the C-terminus. This side reaction cannot be suppressed with the popular triisopropylsilane (TIS) cracking additive alone. In fact, when TFA/TIS/H, the by-products can be the main product2Use the Cocktail OR Neckline. This side reaction can be suppressed by adding ethylene dithiol (EDT).
use
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