DNA display III. Solid-phase organic synthesis on unprotected DNA

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.     

A convenient approach to synthesizing peptide C-terminal N-alkyl amides

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides.     

Determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis

A new method is described for the determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis. The method involves the addition of a second nucleophile to a solid phase coupling reaction at different time intervals and measuring the amount of activated amino acid intercepted. Using the DCCI-mediated coupling reaction of Boc-Ala-OH and H-Gly-O-resin with H-Gly-O-tBu as the second nucleophile, it was determined that $\text{ca}$. 61% of the theoretical maximum amount of activated Boc-Ala-OH was available after 8 hr of reaction.     

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.     

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.     

Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins. Consequently, in order to obtain equimolar peptide mixtures through the use of mixtures of protected amino acids, the ratio of reaction rates of one amino acid over another must be constant irrespective of the resin-bound amino acid. If this premise is true, this method of synthesis offers a significant advantage over the previous method since, theoretically, equimolar peptide libraries could be synthesized. The influence of the resin-bound amino acid on the relative reaction rates of incoming amino acids was investigated in the current study. © 1994 John Wiley & Sons, Inc.     

Kinetically controlled synthesis of dipeptides using ficin as biocatalyst.

The application of the sulfhydryl protease ficin as biocatalyst is proposed as a novel method for enzyme-catalyzed synthesis of dipeptides. The negligible peptidase but considerable esterase activity at alkaline pH facilitated the kinetically controlled formation of peptide bonds by coupling the ester substrates Z-Ala-OMe and Z-Gly-OMe with L-alanine, D-alanine, L-glutamine, D-glutamine and L-Cys(acetamidomethyl) respectively. The reaction is accomplished without the occurrence of secondary peptide hydrolysis. Under optimum reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 4.8% ethanol, 40 degrees C), the peptide yields ranged from 5 to 91%, depending on the structure of the amino and/or carboxyl component. No racemization was observed in the enzymatic reaction. Application of short-chain peptides has been advocated recently in clinical nutrition. Ficin-catalyzed peptide synthesis might be an attractive biotechnological approach for the synthesis of suitable dipeptides in this respect.     

Resin probe analysis. II. Amino acid coupling reactions.

Resin probe analysis has been employed to evaluate the availability of dicyclohexylcarbodiimide (DCC)-activated amino acids, the relationship between coupling time and reaction yield, and the influence of triethylamine (TEA) concentration on peptide bond formation. Results are presented for five amino acids which indicate that the coupling reactions plateau within 5 min, and no significant increase in yield is observed for longer incubation times. Large decreases in coupling yield (70–90%) were observed at concentrations of TEA above 0.01 m. Inactivation appears to be dependent in part upon amino acid structural features. In the absence of TEA, DCC-activated t-butyloxycarbonyl (Boc)-glycine was stable in the activated state for hours. peptide bond formation showed little or no amino acid concentration-dependence in the range of 0.01–0.04 m. Resin probe experiments provide quantitative data on reaction progress and factors that influence the availability and reactivity of activated amino acids.     

Peptide synthesis containing a B-cell and a T-cell epitope on dextran beads and evaluation of humoral response against bead-peptide construct

Summary Cross-linked dextran beads were chemically modified with Fmoc-βAla to give an amino functionalized support suitable for solid-phase peptide synthesis. On this support, a peptide compriising B-cell and T-cell epitopes in tandem was synthesized manually by Fmoc procedure monitoring the coupling reaction by bromophenol blue procedure and standard Kaiser test. The quality of peptide synthesis was verified by RP-HPLC and mass spectrometry. The size and morphologic characteristics of dextran beads were conserved after peptide synthesis. Furthermore, we analyzed immunogenicity in mice of the T-B peptide on dextran beads compared with T-B peptide immunogenicity when administered in CFA/IFA. In both cases, titers were high and there was not a significant difference in antibody titers between groups (p<0.05). But in contrast CFA they are biocompatible and did not induce any adverse reaction at the site of injection. This experiment suggests that the peptides synthesis on dextran beads can be used to raise the immunogenicity of synthetic peptide in vaccines or therapeutics.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

Conformation studies on oligoalanines, substance P, and the myoglobin sequence (66-73). Circular dichroism of polyethyleneglycol-bound peptides]

The conformation of polyethylene glycol-bound peptides, synthesized by the liquid-phase method, was investigated. This marcromolecular C-terminal protecting group is transparent in the visible and the ultraviolet range to 190 nm and solubilizes peptides in many different solvents. The CD spectra of the polymer-bound myoglobin sequence 66–73 and of the biologically active undecapeptide “substance P” were measured in each step of the synthesis. In both examples the formation of a secondary structure during the growth of the peptide chain was found. In the hydrophobic octapeptide containing the myoglobin sequence 66–73, the influence of either the blocked or the free N-terminal amino group on the conformation was observed. The blocked octapeptide in trifluoroethanol showed a higher degree of α-helix contribution than in its free state. The conformation of the polyethylene glycol-bound nona- and decaalanine in trifluoroethanol and water was determined. The peptide with a free amino end group has β-conformation in trifluoroethanol as well as in water. The corresponding N-Boc-protected derivatives show helical structure. The amino end group has a decisive influence on the formation of β-structure. The method of CD investigation of polymer-bound peptide sequences during the peptide synthesis in solution enables one to determine the influence of protecting groups and the chain end of a peptide on its conformation. It is also possible to study the relationship between the secondary structure, the chain length, and the kinetic of the coupling reaction in different solvents. Since the crystallization method for the liquid-phase peptide synthesis allows one to synthesize peptides in very short time, a new method of studying peptide conformations is opened.     

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.     

New branched amino acids for high affinity dendrimeric DC-SIGN ligands

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized.Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido functional groups. As an example of application, d-mannose and l-fucose residues were linked through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with DC-SIGN using SPR competition assay.     

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.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

An apparatus for simultaneous manual solid-phase synthesis of multiple peptide analogs

A new design of reaction vessel for simultaneous manual solid-phase synthesis of multiple peptide analogs is described. Simultaneous use of four of these vessels attached to a single rotary mixer has been successfully applied to synthesis of two sets of four decapeptide amide analogs. Efficient coupling was indicated by chemical determination at the end of each synthesis cycle and overall final yields of between 78 and 84% were obtained. The products obtained were of a high quality, as assessed by high-performance liquid chromatography and amino acid analysis. This system allows expeditious synthesis of multiple peptide analogs for structure-function studies with economical use of efficiently ventilated laboratory space.     

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.     

Enzymatic formation of Glu-Xaa and Asp-Xaa bonds using Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems.

The capability of Glu/Asp-specific endopeptidase from Bacillus licheniformis to form Glu/Asp-Xaa bonds in frozen aqueous systems was investigated. Under frozen state conditions, the enzyme was able to catalyse peptide bond formation more effectively than in liquid reaction mixtures. The acceptance of amino components which were completely inefficient nucleophiles at room temperature indicates a changed specificity of Glu/Asp-specific endopeptidase under frozen state conditions. Protease-catalysed coupling of two acidic amino acids was demonstrated for the first time. The utilization of Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems offers new possibilities in enzyme-catalysed peptide synthesis.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A switchable stapled peptide

The O‐N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence‐containing peptides, cyclic peptides, epimerization‐free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α‐amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O‐N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.