Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

The coupling factor of photophosphorylation and the electric properties of the thylakoid membrane.

The rate of ATP synthesis of illuminated chloroplasts is correlated with the electric conductance of their inner membranes. In agreement with previous studies it is shown that ATP synthesis is paralleled by an increased conductance of the thylakoid membrane. This conductance together with the ability to form ATP is abolished if chloroplasts are treated with an antibody against the coupling factor CF1. It is not influenced by the fragmented monovalent antibody. This parallels the lack of influence of the fragmented antibody on ATP synthesis in contrast to its influence on hydrolysis and exchange reactions. We conclude that there are different sites for the interaction of the coupling factor with adenine nucleotides. Extraction of the coupling factor is shown to increase the membrane conductance by more than two orders of magnitude. Reincorporation of the crude coupling factor partially restores the net conductance of the membrane (increase in resistance by a factor of 2.5), while a higher degree of restoration was observed for ATP synthesis and the proton conductivity of the membrane. We conclude that the extraction procedure opens different conductive channels in the membrane; a proton specific one, possibly associated with the binding protein for the coupling factor, plus other channels for "non-protons" which in contrast to the proton channel cannot be plugged by reincorporation of the coupling factor.     

Synthesis and application of Fmoc-His(3-Bum)-OH.

This paper presents a reevaluation of the synthesis and properties of Fmoc-His(3-Bum)-OH regarding its application in SPPS with minimal racemization of histidine residues during coupling and esterification reactions. By-product formation during the deprotection of the test peptides could be significantly reduced by scavenging the concomitantly formed HCHO, e.g. with methoxyamine.     

In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).     

A new detector for fully automatic peptide synthesis

A new method of monitoring the rate of reactions in solid-phase peptide synthesis is described. A conductivity detector in the reaction cell (patent applied for) enables the deprotection, washing and subsequent coupling stages to be examined in detail. The half-lives of the reactions can be calculated and hence the optimum reaction times can be predicted. Difficult sequences are sensed and appropriate action taken completely automatically.     

Real time monitoring of acylations during solid phase peptide synthesis: a method based on electrochemical detection

Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

Synthesis of peptide sequences related to thrombospondin: factors affecting aspartimide by-product formation.

Aspartimide formation is one of the most common secondary reactions on solid phase peptide synthesis. In the present work, we describe the optimization of the synthesis of two thrombospondin fragments containing an Asp-Gly sequence that show a strong tendency to form cyclic aspartimide derivatives in an unusual high percentage. Several different strategies were applied changing type of resin, Fmoc-deprotection reagents, coupling additives, resin cleavage cocktails and the use of Hmb-Gly derivative to minimize the extension of this byproduct. Best results were obtained with cross-linked ethoxylate acrylate (CLEAR-cross-linked ethoxylate Acrylate Resin)-type resin and pip/dimethylformamide deprotection. Besides, as in biological assays the aspartimide containing sequence resulted to be more active than the linear one, the optimization of its synthesis was also carried out.     

Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.     

A new detector for fully automatic peptide synthesis

A new method of monitoring the rate of reactions in solid phase peptide synthesis is described. A conductivity detector in the reaction cell enables the deprotection, washing, and subsequent coupling stages to be examined in detail. The half lives of the reactions can be calculated and hence the optimum reaction times predicted. The aggregation of peptide chains and subsequent collapse of the resin is observed. Difficult sequences are sensed and appropriate action taken completely automatically.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.     

Synthesis of Oligodeoxynucleoside Phosphoro-Monothioates and Phosphorodithioates by a Phosphotriester Method

Abstract

A phosphotriester method for the synthesis of dithymidine phosphoromonothoates and phosphorodithioates with new S-protecting groups has been investigated. Four of the S-protecting groups possesed catalytic activity, however side reactions occurred during deprotection. The best S-protecting group was 4-chloro-2-nitlobenzyl which could be removed with a minimum of side reactions (0.3 %). The coupling reagent PyFNOP (14) gave protected dithymidine phosphoromonothioate in 96 % yield after 15 min coupling. Furthermore PyFNOP chemoselectively activates oxygen in nucleoside phosphorodithioate monomers 9 and can be used for the synthesis of oligodeoxynucleoside phosphorodithioates with mixed base sequences.     

The coupling factor of photophosphorylation and the electric properties of the thylakoid membrane

The rate of ATP synthesis of illuminated chloroplasts is correlated with the electric conductance of their inner membranes. In agreement with previous studies it is shown that ATP synthesis is paralleled by an increased conductance of the thylakoid membrane. This conductance together with the ability to form ATP is abolished if chloroplasts are treated with an antibody against the coupling factor CF₁. It is not influenced by the fragmented monovalent antibody. This parallels the lack of influence of the fragmented antibody on ATP synthesis in contrast to its influence on hydrolysis and exchange reactions. We conclude that there are different sites for the interaction of the coupling factor with adenine nucleotides.Extraction of the coupling factor is shown to increase the membrane conductance by more than two orders of magnitude. Reincorporation of the crude coupling factor partially restores the net conductance of the membrane (increase in resistance by a factor of 2.5), while a higher degree of restoration was observed for ATP synthesis and the proton conductivity of the membrane. We conclude that the extraction procedure opens different conductive channels in the membrane; a proton specific one, possibly associated with the binding protein for the coupling factor, plus other channels for “non-protons” which in contrast to the proton channel cannot be plugged by reincorporation of the coupling factor.     

Solution Phase Synthesis of Dithymidine Phosphorothioate by a Phosphotriester Method Using New S-Protecting Groups

Abstract

ABSTRACT

A phosphotriester method for the synthesis of dithymidine phosphorothioates with eight S-protecting groups has been investigated. Three of the S-protecting groups possesed catalytic activity, however side reactions occurred under deprotection. The best S-protecting group was 4-chloro-2-nitrobenzyl which could be removed with a minimum of side reactions (0.3 %). The coupling reagent PyFNOP (11) gave protected dithymidine phosphorothioate in 96% yield after 15 min coupling.     

A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.     

A convenient solid-phase method for synthesis of 3'-conjugates of oligonucleotides.

We present a new procedure for the preparation of 3'-conjugates of oligonucleotides through solid-phase synthesis. A suitable universal solid support was readily prepared using a series of peptide-like coupling reactions to incorporate first a spacer and then an L-homoserine branching unit. The N-alpha-position of the homoserine carries an Fmoc protecting group that is removed by treatment with piperidine to liberate an amino group suitable for attachment of the conjugate (e.g., small organic molecule, fluorescent group, cholesterol, biotin, amino acid, etc.) or for assembly of a short peptide. The side-chain hydroxyl group of the homoserine carries a trityl protecting group. After TFA deprotection, the hydroxyl group acts as the site for oligonucleotide assembly. An additional spacer, such as aminohexanoyl, may be incorporated easily between the conjugate molecule and the oligonucleotide. A number of examples of synthesis of 3'-conjugates of oligonucleotides and their analogues are described that involve standard automated oligonucleotide assembly and use of commercially available materials. The linkage between oligonucleotide and 3'-conjugate is chirally pure and is stable to conventional ammonia treatment used for oligonucleotide deprotection and release from the solid support. The homoserine-functionalized solid support system represents a simple and universal route to 3'-conjugates of oligonucleotides and their derivatives.     

RNA synthesis using a universal, base-stable allyl linker.

The application of a universal allyl linker, 9-O-(4,4'-dimethoxytrityl)-10-undecenoic acid, to the solid phase synthesis of RNA molecules is described. Use of this linker simplifies significantly the isolation and purification steps in RNA synthesis. The linker is universal in that it does not contain a nucleoside. The 3'terminal nucleoside is instead attached to the support in the first coupling step. The resultant RNA fragment is then obtained as the 3'-phosphate. The linker is base-stable, and thus all reagents used during deprotection can simply be washed away, leaving the RNA attached. Further, tritylated short fragments resulting from chain cleavage for any reason are also washed away before separation from the support. This linker is compatible with any current synthetic methodology and any amino functionalized support. Of course, silica supports would not be compatible with fluoride reagents. It could also be used to advantage for other applications. Because it is cleaved under conditions orthogonal to those used during many common reactions, the range of post-synthetic manipulations that can be carried out without cleavage from the support is extended significantly.     

Synthesis and Evaluation of Oligonucleotides of High Purity

Abstract

We have developed and evaluated methods for the production of highly pure oligonucleotides.

Presently the solid phase synthesis in an automated DNA synthesiser applying the phosphoramidite chemistry can be regarded as a standard. During the synthesis several undesirable by-products arise:

- incomplete coupling (1%) leads to 5′-truncated sequences. These sequences are acetylated at their 5′-hydroxyl group to prevent further elongation in subsequent coupling steps, but this “capping step” is incomplete, the capping-yield is 90%, leading to accumulation of sequences of the length n-1 with internal deletions.

- the glycosidic bond to N-protected purines, especially adenine, is susceptible to acid leading to depurination and subsequently to strand scission during alkaline deprotection of the oligonucleotide. This gives rise to 3′- and to 5′-truncated sequences. The 3′-truncated sequences will not be removed by standard Rp HPLC as they are tritylated.

- the reactions involved in synthesis and deprotection may cause base modifications (full length product with damaged bases).

- insufficient deprotection procedures may result in incomplete removal of protecting groups, especially from the bases (full length products with altered bases).

We have set up two different schemes (Fig. 1 and Fig. 2) for synthesis and purification, which should provide highly pure oligonucleotides with the potential of adapting to large scale production:

- accumulation of n-1 sequences (failure of capping) will be avoided by a double capping procedure using phosphite in the first capping step and an acetic anhydride capping reagent in the second capping step, as described in the literature¹.

- 3′-truncated sequences are removed by different methqds in the two schemes. In scheme I (Fig. 1) the 3′-truncated sequences can be washed off, as the 3′-full length product still is anchored to the solid support after deprotection. In scheme II (Fig. 2) the 3′truncated sequences are digested by snake venom phosphodiesterase. The 3′-full length product is protected against digestion by a 3′ - 3′-inverted end. An oligo with a correct 3′-end is, in both schemes, eventually obtained by cleaving with RNase between the ribo unit and the requested DNA-sequence.

- 5′-truncated sequences are removed by Rp HPLC using the DMTr group of the last coupling step (trityl-on synthesis) as a hydrophobic tag.

Very labile protecting groups will be used to avoid problems with deprotection.     

Ninhydrin as a reversible protecting group of amino-terminal cysteine.

The proximity of the alpha-amine and beta-thiol of alpha-amino terminal-cysteine (NT-Cys) residues in peptides imparts unique chemical properties that have been exploited for inter- and intra-molecular ligation of unprotected peptides obtained through both synthetic and biological means. A reversible protecting group orthogonal to other protection strategies and reversible under mild conditions would be useful in simplifying the synthesis, cleavage, purification and handling of such NT-Cys peptides. It could also be useful for the sequential ligation of peptides. To this end, we explored tri-one chemistry and found that ninhydrin (indane-1,2,3 trione) reacted readily with cysteine or an NT-Cys-containing peptide on- or off-resin at pH 2-5 to form Ninhydrin-protected Cys (Nin-Cys) as a thiazolidine (Thz). The Thz ring, protecting both the amino and thiol groups in Nin-Cys, completely avoids the formylation and Thz side reactions found during hydrofluoric acid (HF) cleavage when N-pi-benzyloxymethyl histidine groups are present. Nin-Cys is stable during coupling reactions and various cleavage conditions with trifluoroacetic acid or HF, but is deprotected under thiolytic or reducing conditions. These properties enable a facile one-step deprotection and end-to-end-cyclization reaction of Nin-Cys peptides containing C-terminal thioesters.     

In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (>0.2 m) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the _.base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1–50,Cys.amide), HIV-1 protease(53–99), and the full length HIV-l protease(1–99). Republished with the permission of Blackwell Publishing from International Journal of Peptide Protein Research, volume 40, pp. 180–193, 1992. A preliminary account of this work was presented at the 12th American Peptide Symposium, Cambridge, MA, July 16–21, 1991 (ref. 43). Dedicated to Professor Bruce Merrifield on the occasion of his 70th birthday.