Solid‐phase synthesis of C‐terminally modified peptides

Biochimica et Biophysica Acta (BBA) - General Subjects, 2000

A new method is described for the selective`in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined A-amino group of a lysine residue. The K-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially-available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective`in synthesis' labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported.

Journal of Peptide Science, 1996

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modillcations including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C-and/or N-terminals. After incubation with living cultured cells these two probes were locallzed and it is concluded that the post-synthesis modillcations can strongly modify the localization of the peptide.

Amino Acids, 2014

Here we review the strategies for the solidphase synthesis of peptides starting from the side chain of the C-terminal amino acid. Furthermore, we provide experimental data to support that C-terminal and side-chain syntheses give similar results in terms of purity. However, the stability of the two bonds that anchor the peptide to the polymer may determine the overall yield and this should be considered for the large-scale production of peptides. In addition, resins/linkers which do not subject to side reactions can be preferred for some peptides.

Organic & Biomolecular Chemistry, 2020

A go-to compilation of recent strategies to access C-terminally modified peptides contextualized by a discussion of the major synthetic challenges that have historically hampered progress in this area.

The Journal of Organic Chemistry, 1994

Two novel handles for peptide amide preparation under mild conditions were developed for use in highly efficient solid-phase peptide synthesis. These handles, 5-{ [(R,S)-5-[(9-fluorenylmethoxy-carbony1)aminol-l0,l l-dihydrodibenzo[u,dlcyclohepten-2-ylloxy}valeric acid (CHA) and 5-{ [(R,S)-5-[(9-fluorenylmethoxycarbonyl)aminoldibenzo[u,dlcyclohepten-2-ylloxy}vale~c acid (CHE), were attached to the solid support and were used for syntheses of peptides having a C-terminal amide by the fluorenylmethoxycarbonyl strategy. The cleavability of CHA and CHE was determined and compared with the that commercially available amide handles. CHA and CHE handles can be rapidly cleaved from the polymer support without significant side reactions using lower acid concentrations than those required for conventional handles. As CHA can be easily synthesized in large amounts, it is suitable for peptide amide preparation for pharmaceuticals. As CHE can be cleaved at very low concentrations of acid, it is especially suitable for preparing side chainprotected peptide amides. Several brain-gut peptides having a C-terminal amide were synthesized in high yield and high purity with these novel handles.

FEBS Letters, 1977

Analytical Biochemistry, 1996

of peptides is also studied to reproduce specific determi-Recent developments in allyl chemistry and pallanants of high-molecular-weight proteins. The length of dium solubilization allow automated continuous-flow the peptides used for these studies is in general limited solid-phase synthesis of cyclic or branched peptides, (up to 15 amino acids) to overcome the problems that with specific side-chain cleavage and on-line cyclizaarise with increasing length of the peptide. However, tion. In this paper, we adapted the method to the synthese peptides show a higher flexibility compared to thesis of cyclic peptides bearing an anchoring tail on the native protein. For this reason, their biological aca side chain of the cycle. Side products were obtained tivity is usually lower than in the native conformation with the standard procedure and an additional washunless they are in some way rigidified. One way to ing step had to be introduced in the synthesis protocol achieve this is to synthesize different head-to-tail cyclic to remove side products resulting from the palladium peptides with restricted conformational flexibility and allyl cleavage step. The method is illustrated by the to determine which of these analogs retains biological automated synthesis of cyclo[-DVal-Arg-Gly-Asp-Glu activity (1-5). (-eAhx-Cys-NH 2)-] which contains the Arg-Gly-Asp ad-Series of cyclic peptides have been used to study the hesion motif (RGD) recognized by cellular integrins. conformation-activity relationship of the Arg-Gly-Asp The tail of the peptide was designed with a thiol at the (RGD) 3 sequence (6-9), which is found in many procarboxylic end to allow subsequent grafting by covateins of the extracellular matrix like fibronectin, collalent attachment. Such tailed cyclic peptides can be gens, laminin, or tenascin (10) but also in dysintegrins grafted on different supports for new applications in like kistrin (11) or echistatin (12) present in venoms. biomaterial design, cell adhesion assays, affinity chro-This RGD sequence has been shown to be specifically matography, immunization, vaccine development, recognized by cell membrane receptors known as inte-ELISA kits, and the building of libraries of conformationally constrained peptides.

The Journal of Organic Chemistry, 1991

Bioconjugate Chemistry, 2006

Journal of Peptide Science, 2000

A new aminoethyl-polystyrene linker, stable at low concentrations of TFA, has been developed for the solid phase synthesis of peptide amides. The described linker is stable under conditions which remove Bu t protecting groups (30-50% TFA in DCM) and the desired product can be finally cleaved off the solid support in 95% TFA (5% H 2 O). Model peptide amides and other N-alkylated peptide amides have been successfully synthesized in good yield and purity.

The Journal of Organic Chemistry, 1996

[[9-[(9-Fluorenylmethyloxycarbonyl)amino]xanthen-2(or 3)-yl]oxy]alkanoic acid (XAL) handles have been prepared by efficient four-step routes from 2-or 3-hydroxyxanthone and coupled onto a range of amino-functionalized supports. The resultant XAL supports are the starting points for solidphase peptide synthesis by Fmoc chemistry. Upon completion of chain assembly, C-terminal peptide amides are released in excellent yields and purities by use of low concentrations [1-5% (v/v)] of trifluoroacetic acid (TFA) in dichloromethane, often without a need for added carbocation scavengers. These cleavage conditions allow retention of all or a significant portion of tert-butyl type and related side-chain protecting groups, which subsequently may be removed fully in a solution process carried out at higher acid concentration. XAL supports are particularly useful for the synthesis of acidsensitive peptides, including tryptophan-containing sequences that are known to be susceptible to yield-and/or purity-reducing alkylation side reactions. The effectiveness of this chemistry was shown with the syntheses of prothrombin (1-9), acyl carrier protein (65-74), Tabanus atratus adipokinetic hormone, fragments of the protein RHK 1, CCK-8 sulfate, and oxytocin. Furthermore, the application of XAL supports for the preparation of fully protected peptide amides has been demonstrated. Work from our laboratories 6,7 and others 8 has provided several methods for the stepwise solid-phase synthesis of C-terminal peptide amides in conjunction with protection schemes using the base-labile N R-9-fluorenylmethyloxycarbonyl (Fmoc) function. 9 The best approaches use appropriate handles, 10 which are in effect N-protected (aromatic) amino acids that can be coupled onto aminofunctionalized supports and later serve as a starting point

European Journal of Nuclear Medicine and Molecular Imaging, 2002

A rapid method for radiolabelling short peptides with 18 F (t 1/2 =109.7 min) for use in positron emission tomography (PET) was developed. Linear peptides (13mers) were synthesised using solid phase peptide synthesis and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. The peptides were assembled on a solid-phase polyethylene glycol-polystyrene support using the "hyper acid labile" linker xanthen-2-oxyvaleric acid and were labelled in situ with 4-[ 19 F]-or 4-[ 18 F]fluorobenzoic acid. Optimum coupling of 4-[ 19 F]fluorobenzoic acid to the peptidyl resin was achieved within 2 min using N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HATU/DIPEA), and optimum cleavage was achieved within 7 min using trifluoroacetic acid/phenol/water/Triisopropylsilane at 37°C. The linear peptides were rapidly labelled with 4-[ 18 F]fluorobenzoic acid with an overall radiochemical yield of 80%-90% (decay corrected), a radiochemical purity of >95% without HPLC purification and an overall synthesis time of 20 min. This novel method was used to label peptides containing the arginine-glycine-aspartic acid (RGD) motif, the binding site of many integrins. In vitro studies showed that the fluorobenzoyl prosthetic group had no deleterious effect on the ability of these peptides to inhibit the binding of human cells via integrins. Biodistribution studies in tumour-bearing mice showed that although the linear peptides were rapidly removed from the circulation by the liver and kidneys, there was a transient and non-RGD-dependent accumulation in the tumour of both the test and the control peptides. The use of more selective peptides with a longer half-life in the circulation combined with this rapid labelling technique will significantly enhance the application of peptides in PET.

Journal of Peptide Research, 1999

Reissmann, S. Synthesis of N-carboxyalkyl and Naminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides. Abstract: To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH 4 . N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing ef®cient puri®cation by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or¯uorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.

ACS Sustainable Chemistry & Engineering, 2019

mixture (2 × 3 mL each). A solution of Fmoc-Leu-OH (3 equiv), N,N′-diisopropylcarbodiimide (DIC) (3 equiv), and Oxyma Pure (3 equiv) in the proper mixture, preactivated for 5 min, was charged onto the resin and stirred for 1 h. After the peptide coupling, the resin was washed with DMF, DCM and DMF or mixture, iPrOH, and mixture (2 × 3 mL each). Then, 20% piperidine in DMF or selected mixture was charged on the resin (2 × 3 mL × 15 min). The resin was washed and ready for the subsequent couplings, deprotections, and washings, as reported before, to obtain the pentapeptide. The peptide was cleaved from the resin with trifluoroacetic acid (TFA)/H 2 O/ triisopropylsilane (TIS) (95:2.5:2.5) solution for 2 h at room temperature. The crude was directly analyzed by HPLC-MS. Solid-Phase Synthesis of H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (Linear Octreotide) in 70:30 Anisole/Dimethyl Carbonate (Method 8). The synthesis was performed in a glass syringe, attached at the bottom to a vacuum source to remove excess of reagents and solvents. The resin (H-Thr(tBu)-ol-2CT-PS 0.6 mmol/g, 500 mg) was washed with 3 mL of Mix C3, 3 mL of iPrOH, and 3 mL of Mix C3. A preactivated solution of Fmoc-Cys(Trt)-OH (3 equiv), DIC (3 equiv), and Oxyma Pure (3 equiv) in Mix C3 (3.3 mL) was charged onto the resin and stirred for 1 h. After the peptide coupling, the resin was washed with 3 mL of Mix C3, 3 mL of iPrOH, and 3 mL of Mix C3. Fmoc removal was performed by adding 2 × 3 mL of 20% piperidine in Mix C3 on the resin, shaking it for 10 min each. After the deprotection, the resin was washed with 4 × 3 mL of Mix C3. The resin was ready for the subsequent couplings, deprotections, and washings, as reported before, to obtain the decapeptide. The final washings were performed with Mix C3 (4 × 3 mL) and iPrOH (3 × 2 mL). The peptide was cleaved from the resin with TFA/TIS/1-dodecanthiol (9 mL/0.7 mL/0.6 mL) solution for 4 h at room temperature. The solution was recovered by filtration. Diisopropyl ether (37 mL) was added dropwise at 0−5°C to the acidic solution until precipitation of peptide was achieved. The resulting mixture was stirred for 1.5 h at 0−5°C. The precipitate was filtered, washed with diisopropyl ether and petroleum ether, and dried under vacuum, affording an off-white solid. The crude was analyzed by HPLC-MS. For the synthesis with method 1, as a substitution for Mix C3 and iPrOH, DMF and DCM were used. Cyclization of Linear Octreotide. Crude trifluoroacetate linear Octreotide (1 g of a raw synthetic product containing 82.3% or 88.0%

Amino Acids, 2013

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N 0-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N a-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N a-(9H-fluorenyl-9-methoxycarbonyl)-N b ,N b0-bis[2-(tert-butoxycarbonylamino)ethyl]-L-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity. Keywords N-alkylation Á Reductive amination Á One-pot procedure Á Branched basic amino acids Á Solid-phase peptide synthesis Á Indolicidin