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Development and characterisation of protecting groups that enhance the solubility of synthetic peptides: Methods to improve the aqueous solubility of hydrophobic peptides
Stockholm University, Faculty of Science, Department of Neurochemistry. (Anders Undén )
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chemical synthesis of peptides is generally performed by solid phase peptide synthesis (SPPS). Although SPPS is a rapid and convenient method to prepare peptides, the major methodological problem in SPPS is related to solvation/solubility. Poor solvation of the peptide during the synthesis on solid phase will frequently lead to deletion peptides and if the peptide is poorly soluble after it has been cleaved from the resin, it can be difficult to purify by liquid chromatography. In order to increase the solubility of peptides, two new amino acid derivatives and a modification of the 2-hydroxy-4-methoxy-benzyl (Hmb) group, a protecting group for the peptide bond, have been developed. The Boc-N-methyl-N-[(2-methylamino)ethyl]carbamoyl (Boc-Nmec) group was used for protection of aromatic hydroxyl groups of tyrosine and the Hmb group. The Boc-4-(N-methyl-amino)butanoyl (Boc-Nmbu) group was used for protection of the indole nitrogen on tryptophan. The new derivatives were introduced into model peptides by standard protocol for Fmoc chemistry. The Boc protection of the Nmec/Nmbu group is removed during the cleavage of the peptide from the resin but the Nmec/Nmbu group is still attached to the peptide. The Nmec/Nmbu group contains a secondary amino group that is protonated at low pH and thereby increases the solubility during purification and handling of the peptide in aqueous solutions. By raising the pH the Nmec (pH 8)/Nmbu (pH 9) group is cleaved by an intramolecular cyclization reaction to give the fully deprotected peptide. Amyloid b (1-42) is a large peptide that is very difficult both to assemble on solid phase and to purify by HPLC.  It is shown that by using the Boc-Nmec protected dipeptides, amyloid b (1-42) could be synthesized by SPPS with only traces of by-products and that the peptide was easy to purify by HPLC. It is likely that the new protecting groups would be very useful in synthesis and handling of hydrophobic peptides.

 

 

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2009. , 69 p.
Keyword [en]
Solid phase peptide synthesis, Fmoc chemistry, solubility problems, tryptophan, tyrosine, Nmec, Nmbu
National Category
Organic Chemistry
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-27202ISBN: 978-91-7155-867-1 (print)OAI: oai:DiVA.org:su-27202DiVA: diva2:212835
Public defence
2009-05-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2009-05-08 Created: 2009-04-24 Last updated: 2015-03-16Bibliographically approved
List of papers
1. A carbamoyl-protective group for tyrosine that facilitatespurification of hydrophobic synthetic peptides
Open this publication in new window or tab >>A carbamoyl-protective group for tyrosine that facilitatespurification of hydrophobic synthetic peptides
2008 (English)In: Tetrahedron Letters, ISSN 0040-4039, Vol. 49, no 23, 3779-3781 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
Dorchester: Elsevier, 2008
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-27550 (URN)10.1016/j.tetlet.2008.04.014 (DOI)000256378800017 ()
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2015-03-16Bibliographically approved
2. Synthesis and purification of aggregation-prone hydrophobicpeptides by the incorporation of an Fmoc dipeptide withthe peptide bond protected with a modified 2-hydroxy-4-methoxybenzyl (Hmb) group
Open this publication in new window or tab >>Synthesis and purification of aggregation-prone hydrophobicpeptides by the incorporation of an Fmoc dipeptide withthe peptide bond protected with a modified 2-hydroxy-4-methoxybenzyl (Hmb) group
2008 (English)In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 49, no 24, 3921-3924 p.Article in journal (Refereed) Published
Abstract [en]

The dipeptide Fmoc-Val-(2-hydroxy-4-methoxybenzyl)Gly-OBzl was synthesized and the 2-hydroxyl group carbamoylated to give a Boc-N(CH(3))CH(2)CH(2)N(CH(3))CO-, (Boc-Nmec-) modification of the 2-hydroxy-4-methoxybenzyl (Hmb) group. After catalytic hydrogenation and purification, the resulting dipeptide Fmoc-Val-(Boc-Nmec-Hmb)Gly-OH was used in solid phase peptide synthesis. During treatment with TFA, the peptide was released from the resin and the Boc group cleaved. The peptide could then be purified with an alkylated peptide bond carrying a cationic charge that both increased the solubility of the peptide during the purification steps and facilitated analysis by MALDI-TOF mass spectrometry. The Nmec group was cleaved by intramolecular cyclization under slightly alkaline conditions, followed by cleavage of the Hmb group by TFA to give the fully deprotected peptide.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-27553 (URN)10.1016/j.tetlet.2008.04.055 (DOI)000256500500021 ()
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2015-03-16Bibliographically approved
3. Synthesis of Amyloid β (1-42) with protected peptide bonds
Open this publication in new window or tab >>Synthesis of Amyloid β (1-42) with protected peptide bonds
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-27555 (URN)
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2016-01-29Bibliographically approved
4. A new protecting group for tryptophan in solid-phase peptide synthesis which protectsagainst acid-catalyzed side reactions and facilitates purification by HPLC
Open this publication in new window or tab >>A new protecting group for tryptophan in solid-phase peptide synthesis which protectsagainst acid-catalyzed side reactions and facilitates purification by HPLC
2009 (English)In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 50, no 24, 2976-2978 p.Article in journal (Refereed) Published
Abstract [en]

The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis (SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group, which improves the solubility of the peptide during HPLC purification. On treatment of the purified peptide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully deprotected peptide and N-methylpyrrolidone.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-27554 (URN)10.1016/j.tetlet.2009.04.014 (DOI)000266188300036 ()
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2015-03-16Bibliographically approved

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