N-Fmoc-a-sulfo-b-alanine: a versatile building block for the water solubilisation of chromophores and fluorophores by solid-phase strategy
Anthony Romieu, Thomas Bruckdorfer, Guillaume Clavé, Virgile Grandclaude, Cédrik Massif and Pierre-Yves Renard
Org. Biomol. Chem. 2011; 9: 5337. DOI: 10.1039/c1ob05730h
An easy and efficient solid-phase synthesis strategy to obtain rapidly water-soluble chromophores/fluorophores in highly pure form has been developed. This first successful use of N-Fmoc-alpha-sulfo-beta-alanine as a SPPS building block opens the way to the future development of promising direct “on-resin” peptide labeling and water-solubilizing methods.
Understanding enzyme immobilisation
Ulf Hanefeld, Lucia Gardossi and Edmond Magner
Chem. Soc. Rev. 2009; 38: 453–468; DOI: 10.1039/b711564b
Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations.
At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical–chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.
Shorter Arginine homologues to stabilize peptides towards tryptic digestion
Petra Henklein, Thomas Bruckdorfer
Chemistry Today 2008; 26(6): 12-15.
Trypsin digests peptides at the position of arginine. Because shorter homologues of arginine with appropriate protecting groups for conventional Fmoc/tBu peptide synthesis are now available, three model peptides containing arginine and two shorter homologues of arginine were synthesized. They were incubated with trypsin in order to explore how stable the corresponding peptides are towards enzymatic degradation. It could be demonstrated that a peptide gains significant stability if arginine is being exchanged by a homologue containing one methylene group less. If arginine is substituted by a homologue with two methylene groups less the model peptide was almost fully stable over 24h towards enzymatic degradation.
A Three-Dimensional Quanititative Structure-Activity Relationship (3D-QSAR) Model for Predicting the Enantioselectivity of Candida antarctica Lipase B
Paolo Braiuca, Knapic Lorena, Valerio Ferrario, Cynthia Ebert, and Lucia Gardossi
Adv. Synth. Catal. 2009; 351: 1293 – 1302
Computational techniques involving molecular modeling coupled with multivariate statistical analysis were used to evaluate and predict quantitatively the enantioselectivity of lipase B from Candida antarctica (CALB). In order to allow the mathematical and statistical processing of the experimental data largely available in the literature (namely enantiomeric ratio E), a novel class of GRID-based molecular descriptors was developed (differential molecular
interaction fields or DMIFs). These descriptors proved to be efficient in providing the structural information needed for computing the regression model. Multivariate statistical methods based on PLS (partial least square – projection to latent structures), were used for the analysis of data available from the literature and for the construction of the
first three-dimensional quanititative structure-activity relationship (3D-QSAR) model able to predict the enantioselectivity of CALB. Our results indicate that the model is statistically robust and predictive.
PEGylation - The Magic Wand IRIS Biotech GmbH - Turning Proteins and other Biopharmaceuticals into Super Performing Block Busters.
PharManufacturing 2007; 34-41
In 2006 the market of modern biopharmaceuticals has reached a volume of over $40.3 billion in USA and over $45 billion world wide (IMS Health, Inc.). It is projected to grow to an annual value of some $100 billion within the next 5 years. The big advantage of proteins, antibodies, siRNA, and other natural products in their usage as drugs is their high specifity in combination with their low side effects. They normally interact with the dedicated target only, and thus do not have activities at any other place in the body. Modern biopharmaceuticals are ideal drugs, however, their significant drawback is their low stability under physiological conditions. Due to the fact that they are similar to biological components, they are easily attacked by the immune system of the body, i.e. by antibodies and proteolytic degradation enzymes. Many efforts have been made by highly sophisticated formulation techniques, special application methods (depots) and chemical modification to improve their pharmacokinetic properties. One recent approach, which shows much better results than other methods tried in the past, is PEGylation, i.e. attaching PolyEthylene Glycol chains (PEG) to the active component. The simplest possibility of PEGylation is attaching a monofunctional PEG chain to a protein, antibody or small drug molecule. Using bifunctional PEGs a link between two compounds can be formed, in order to build dimers or more complex conjugates. Many highly sophisticated compositions are under development and already published.
Preparation of D-amino acids by enzymatic kinetic resolution using a mutant of penicillin-G acylase from E. coli
Chiara Carboni, Hans G. T. Kierkels, Lucia Gardossi, Kamil Tamiola, Dick B. Janssen and Peter J. L. M. Quaedflieg
Tetrahedron Asymmetry 2006; 17: 245–251
We have demonstrated for the first time that D-glutamine (D-Gln) and D-glutamic acid (D-Glu) can be efficiently obtained in high ee (97% and 90%, respectively) by enzymatic kinetic resolution of D,L-Gln and D,L-Glu. This was achieved by enantioselective conversion of the L-enantiomers to their N-phenylacetyl derivatives in aqueous solution, using a mutant of penicillin-G acylase (PGA) from E. coli and phenylacetic acid methylester as the acyl donor. Kinetic modeling studies suggest that the high ee values obtained are both due to a strong enantiopreference for the L-amino acid in the deacylation step of the covalent enzyme intermediate, as well as to completeness of conversion that is transiently obtained as a result of the distinct preference of the mutant PGA for phenylacetic acid methylester over the N-phenylacetyl-L-amino acid product. For the other amino acids tested (Asn, Asp, and Ser), the highest ee values that were obtained for the remaining D-enantiomer are moderate (50–80%) because of lower enantioselectivity in the enzyme deacylation step and due to less complete conversion of the L-amino acid caused by competition for the active site between the acyl donor and the N-phenylacetyl-L-amino acid that is produced. The results demonstrate that the mutated PGA has great potential for the production of optically active D-amino acids by kinetic resolution.
Hexafluoroacetone as Protecting and Activating Reagent: New Routes to Amino, Hydroxy, and Mercapto Acids and Their Application for Peptide and Glyco- and Depsipeptide Modification
Jan Spengler, Christoph Böttcher, Fernando Albericio, and Klaus Burger
Chem. Rev. 2006; 106: 4728-4746
A considerable number of biologically active naturally occurring products are peptides, depsipeptides, and peptide conjugates. Peptide modification can be performed by backbone modification and modification of the substituent pattern of the side-chain of the monomers. Direct therapeutic applications of native peptides are limited. However, major drawbacks of peptide drugs, like low selectivity, rapid degradation by proteases, low lipophilicity, and lack of transport systems to direct peptides into cells, can be overcome by incorporation of new types of monomers into strategic positions. This concept is based on an efficient access to monomers with tailor-made side-chains of readily available R-amino and R-hydroxy acids with HFA (hydroxyfluoroacetone) as bidentate protecting reagent. If multifunctional amino acids are involved, several protection and deprotection steps are usually required. A bidentate protecting/activating concept meets this synthetic challenge by reducing the number of steps if protection of the R-functionality and activation of the adjacent 1-carboxy group as well as subsequent coupling and deprotection of the R-functionality occur simultaneously. Hexachloroacetone and 1,1,1-trichloro-3,3,3-trifluoroacetone react with the R-amino group of amino acids with chloroform elimination to give the corresponding N-trichloroacetyl and N-trifluoroacetyl amino acids, respectively. In contrast, hexafluoroacetone (HFA) forms 2,2-bis(trifluoromethyl)-1,3-oxazolidin-5-ones. Remarkably, no additional R-amino protection is required. Furthermore, HFA was also found to react readily with R-hydroxy and R-mercapto acids to give five-membered heterocycles. In a single step the R-placed functionality and R-carboxy group are protected. The lactone ring represents an activated ester and can be cleaved by various O- and N-nucleophiles in solution and on solid phase to give the corresponding unprotected derivatives in one step. HFA protection occurs site selectively in the presence of unprotected side-chain functionalities, like the α-carboxy group of Asp.
From Production of Peptides in Milligram Amounts for Research to Multi-Tons Quantities for Drugs of the Future
Thomas Bruckdorfer, Oleg Marder and Fernando Albericio
Current Pharmaceutical Biotechnology 2004; 5: 29-43 29
Peptides are key to modern drug discovery. This article reviews the requirements for bulk production of peptides and how it affects research and production of smaller scales. Peptides, as modern drugs, are currently produced in millions in mg-scale for research purpose, in order to better understand the function of biological systems. Some newly discovered sequences form the basis of modern drugs and are now produced in multi-tons. The most popular example is the T-20 peptide (Fuzeon), which is the first peptide produced at such scale by a combination of solid phase and solution phase methodologies. This particular peptide sequence has the ability to dock on the surface of the HIV virus and block the virus from entering into a human blood cell, helping patient life conditions. A multi-ton scale production was made necessary based on the high number of patients, the socio-economical importance of the disease and the strong support by governmental institutions such as the FDA. Fuzeon is the first peptide-based drug that is produced in multi-tons on solid support. This had revolutionary effects on the whole peptide synthesis techniques in general including the production of the starting materials. It also had a positive impact on the cost-effectiveness of peptides for research, as the standard technique for producing peptides in research quantities is solid phase chemistry. The decrease of the cost of all starting materials will lead to an increase of the number of produced peptides, which will certainly bring new interesting and effective sequences to be used as novel drugs.
Resin-bound mercapto acids: synthesis and application.
Mourtas, Spyros; Gatos, Dimitrios; Karavoltsos, Manolis; Katakalou, Christina; Barlos, Kleomenis.
Tetrahedron Letters 2002; 43(18): 3419-3421.
Mercapto acids were attached through their thiol group onto 2-chlorotrityl (Clt)-, trityl (Trt)-, 4-methyltrityl (Mtt)-, 4-methoxytrityl (Mmt)-, and 4,4'-dimethoxytrityl (Dmt)-resins. The new resins were used in the solid-phase synthesis of small mercaptoacylamino alcs. Cleavage from the resins was performed by treatment with trifluoroacetic acid (TFA) solutions in dichloromethane (DCM) using triethylsilane (TES) as scavenger.
Phase change during peptide synthesis. Application of Fmoc-Rink Amide Linker on 2-Chlorotrity Resin
Athanassopoulos, Panagiotis; Barlos, Kleomenis; Hatzi, Olga; Gatos, Dimitrios; Tzavara, Chryssoula.
Editor(s): Epton, Roger. Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids - Small Molecule Organic Chemical Diversity, Collected Papers, International Symposium, 4th, Edinburgh, Sept. 12-16, 1995 (1996), Meeting Date 1995, 243-246. Publisher: Mayflower Scientific, Birmingham, UK CODEN: 64ONA9 Conference written in English. CAN 127:66159 AN 1997:412383 CAPLUS (Copyright 2003 ACS)
A symposium report. Protected peptides were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) amino acids and the acid labile 2-chlorotrityl and the multidetachable 2-chlorotrityl-Rink-linker resins. After their cleavage from the resins, the obtained peptides were esterified in solution with 2-chlorotrityl chloride and DIEA. The obtained esters, were deprotected at the N-terminal function, condensed to larger peptides and reesterified on the resin after the selective removal of the chlorotrityl function. In cases where mixtures of protected peptides were used for the esterification with the trityl-resin, a competition was observed among them for the occupation of the most favourable positions of the resin. Mixtures of protected peptides were also used in fragment condensations in order to evaluate the best possible conditions for the preparation of long-chain peptide libraries.
Synthesis of side-chain to side-chain cyclic peptides and oligolysine cores suitable for the solid-phase assembly of MAPs and TASPs.
Aletras A; Barlos K; Gatos D; Koutsogianni S; Mamos P
International Journal of Peptide and Protein Research 1995; 45(5): 488-96.
The Nε-Mtt function of Nα-9-Fluorenylmethoxycarbonyl-Nε-4-methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH] can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid –trifluoroethanol-dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.
Application of the Trt and Fmoc groups for the protection of polyfunctional α-amino acids.
Barlos, Kleomenis; Mamos, Petros; Papaioannou, Dionysios; Patrianakou, Stella; Sanida, Chariklia; Schaefer, Wolfram.
Liebigs Annalen der Chemie 1987; 12: 1025-30.
Ditrityl amino acids Trt-Lys(Trt)-OH (Trt = CPh3), Trt-Orn(Trt)-OH, Trt-Ser(Trt)-OH, and Trt-Hse(Trt)-OH were prepared and used in the synthesis of trityl-protected peptides, e.g., Trt-Ser(Trt)-Phe-NH2. Selective detritylation of the above amino acid derivatives and ditrityl peptides was achieved with 1% CF3CO2H in CH2Cl2. The resulting N-detritylated amino acids were converted into the corresponding Nα-Fmoc (Fmoc = fluorenylmethyloxycarbonyl) derivatives under Schotten-Baumann conditions using Fmoc-Cl.