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.     

Solid-phase synthesis and cyclization of a large branched peptide from IgG Fc with affinity for Fc gammaRI.

A solid phase approach has been used to synthesize a large branched disulphide peptide from IgG Fc, Ac-F-C*-A-K-V-N-N-K-D-L-P-A-P-I-E-K(Ac-E-L-L-G-G-P-S-V-F)-C*-I-NH2. This peptide combines the lower hinge region of IgG and a proximal beta-hairpin loop, both implicated in binding to Fc gammaRI. Solid phase Tl(tfa)3 cyclization of the linear branched peptide resulted in a poor yield of cyclic hinge-loop peptide (11%) most likely due to steric hindrance caused by the branch. However, if addition of the branch was preceded by solid phase Tl(tfa)3 cyclization of the loop, the yield was excellent at 75%. Cyclic hinge-loop peptide was active in displacing IgG2a from Fc gammaRI expressed on monocyte cell lines with an IC50 of 40 microM, whereas the linear form of this peptide was inactive. The Fc hinge-loop peptide demonstrates the potential for a non-mAb high affinity, immunomodulatory ligand for Fc gammaRI.     

Lanthanide Catalyzed Cyclization of Uridine 3'-p-Nitrophenyl Phosphate

Steady state kinetics and (15)N isotope effects have been used to study the cyclization reaction of uridine 3'-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of the 2'-OH group and departure of the leaving group. The transition state is very late, with a very large degree of bond cleavage to the leaving group, which could be due to a large degree of polarization of the P&bond;O bonds by erbium. Copyright 2000 Academic Press.     

Further improvements on the phosphotriester synthesis of deoxyribooligonucleotides and the oligonucleotide directed site-specific mutagenesis of E. coli lipoprotein gene.

Two improvements that greatly enhance the rate of phosphotriester oligonucleotide synthesis are described: 1) use of hindered primary amines, e.g. t-butyl amine for decyanoethylation of oligonucleotide triester intermediates, and 2) a simplified isolation procedure that eliminates the tedious bicarbonate extraction after each condensing reaction. Using the improved procedures, oligonucleotide fragments can be synthesized as rapidly as using solid phase chemistry. The final products are purer than those obtained by solid phase chemistry since each intermediate block is purified by chromatography. The technique has been used to synthesize five oligonucleotide fragments (size 15 to 20) for the purpose of performing guided site-specific mutagenesis on a cloned E. coli lipoprotein gene.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Involvement of NADPH in the cyclization reaction of carotenoid biosynthesis

Cyclic carotenoids, e.g. beta-carotene, are formed by cyclization of an acyclic precursor, lycopene. The gene, crtY, which encodes lycopene beta-cyclase, has a partial sequence characteristic of a pyridine nucleotide binding domain, and NAD(P)H has been reported to be an absolute requirement for the cyclization reaction in vitro. By complementary incubations with lycopene as substrate and with (4R)-[4-(2)H]NADPH in (1)H(2)O or with unlabelled NADPH in (2)H(2)O in the presence of the purified enzyme, it has now been shown that the hydrogen atom introduced at C(2) in the cyclization comes from water and not from NADPH. The previously proposed mechanism involving the initiation of cyclization by H(+) attack at C(2) of the folded acyclic end group of the precursor is thus confirmed. No hydrogen is transferred from NADPH, which is therefore not involved directly in the cyclization reaction, but must play an indirect role, e.g. as an allosteric activator.     

Synthesis of linear and cyclic opioid‐based peptide analogs containing multiple N‐methylated amino acid residues

A series of six novel opioid peptide analogs containing one to three N‐methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid‐phase method. Introduction of two consecutive N‐methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine‐based coupling reagent, 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium toluene‐4‐sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by‐products than observed with the standard coupling reagents such as TBTU or HATU.Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of methodology for the synthesis of cyclic thioether peptide libraries derived from linear tripeptides.

Work was undertaken to examine methodology for the cyclization of linear tripeptides on the solid phase via intramolecular S-alkylation using the Multipin(trade mark) Solid-Phase Peptide Synthesis platform. While previous work had shown that this chemistry could be used to efficiently cyclize linear tetrapeptide libraries, application of this synthetic strategy to the model linear tripeptide sequence Leu-Ser-Lys resulted in significant cyclic dimer formation. Ultimately, it was found that the addition of a large excess of lithium in the form of LiCl to the cyclization solution, significantly reduced cyclic dimer formation affording highly pure crude cyclic monomer. The application of this modified cyclization protocol to the preparation of cyclic peptide libraries was successfully demonstrated with the synthesis of a 20-membered library 4{1-20} based on the linear tripeptide sequence Leu-Xxx-Lys in which the position Xxx was varied with the standard 20 proteogenic residues.     

Investigation of peptide thioester formation via N→Se acyl transfer

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc‐based solid‐phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid‐phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C‐terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C‐terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N‐terminal cysteine and C‐terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Development of a high-throughput method for the determination of pharmacological levels of plasma D-serine

D-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, D-Serine must be administered at high doses to observe clinical effects. This is due in large part to D-Serine undergoing oxidation by D-Serine acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of D-Serine with a DAAO inhibitor has been suggested as a way to lower the dose of D-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma D-Serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure D-Serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor D-Serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate D-Serine from plasma followed by quantitation of D-Serine using the DAAO-catalyzed reaction. Plasma D-Serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.     

Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila

We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction. This was accomplished by selectively removing the 3'-terminal nucleotide by oxidation and beta-elimination; the beta-eliminated IVS thereby is no longer capable of reacting with itself. However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site. We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis of the guanosine binding site involved in cyclization. The results indicate that functional groups on the guanine that are used in a G-C Watson-Crick base pair are important for the cyclization reaction. This is the same result that was obtained for the guanosine binding site involved in splicing [Bass, B. L., & Cech, T. R. (1984) Nature (London) 308, 820-826]. Unlike splicing, however, certain additional nucleotides 5' to the guanosine moiety make significant binding contributions. We conclude that the guanosine binding site in cyclization is similar to, but not identical with, the guanosine binding site in splicing. The same binding interactions used in cyclization could help align the 3' splice site of the rRNA precursor for exon ligation. We also report that the phosphodiester bond at the cyclization site is susceptible to a pH-dependent hydrolysis reaction; the phosphodiester bond is somehow activated toward attack by the 3'hydroxyl of a guanosine molecule or by a hydroxyl ion.     

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

Effect of various acceptors on the rates of the cyclization and chain-shortening of amylose catalyzed by the cyclodextrin glycosyltransferase from Klebsiella pneumoniae M 5 al. Improvement of new photometric assay methods

The effect of various acceptors on the cyclization and chain-shortening reaction of amylose catalyzed by cyclodextrin glycosyltransferase [(1 leads to 4)-alpha-D-glucan: [(1 leads to 4)-alpha-D-glycopyranosyl]transferase (cycling) EC 2.4.1.19] from Klebsiella pneumoniae M 5 al was studied by use of photometric-assay methods. The requirements for the acceptor were the same for both cyclization and chain-shortening, indicating the close relationship between both reactions. Maltose proved to be the most effective (2.48- and 5-fold acceleration of the cyclization and chain-shortening, respectively, in the presence of 584 micrometers maltose). The dependence of the chain-shortening reaction on the conformational state of the amylose molecules is discussed.     

A novel route to synthesize Freidinger lactams by micowave irradiation.

The incorporation of a Freidinger-like lactam structure into the backbone of peptides has been proven to be an useful strategy in the design of a variety of conformationally restricted targets. Several different strategies have been developed toward Freidinger lactams but no one resulted to be completely facile. Here, we report an efficient strategy that involves the iodo-derivatives in side chain of an appropriate amino acid used as electrophilic agent, and the standard solid phase peptide synthesis assisted by microwave irradiation. The methodology developed could be useful to perform Freidinger-like lactams with defined stereochemistry for routine use in solid phase peptide chemistry.     

The synthesis and characterization of 8-methoxy-5'-deoxyadenosylcobalamin: a coenzyme B(12) analog which,following Co-C bond homolysis,avoids cyclization of the 8-methoxy-5'-deoxyadenosyl radical

The compound 8-methoxy-5'-deoxyadenosylcobalamin (8-MeOAdoCbl), has been synthesized in 37% yield and > or = 95% purity by HPLC, monitored at both 254 and 525 nm, or 90+/-2% purity as judged by the (1)H NMR spectrum of the aromatic cobalamin region. This is the first synthesis of this complex in which sufficient details are reported, where a yield and purity are reported, and where key problems in the synthesis and purification are overcome, so that 8-MeOAdoCbl can actually be obtained for use in other studies. Also demonstrated is the clean Co-C bond homolysis of 8-MeOAdoCbl to give initially 8-MeOAdoCbl and Co(II)Cbl in a UV-visible thermolysis experiment at 110 degrees C, results which show that the 8-MeO moiety suppresses the cyclization to the 8,5'-anhydro-adenosine otherwise seen for the adenosyl radical (Ado)*. Suppression of this cyclization pathway makes 8-MeOAdoCbl invaluable for studying the kinetic isotope effect (KIE) of the Ado* plus substrate H* abstraction reaction, a component of the first definitive test of Klinman's hypothesis that the optimization of enzyme catalysis may entail strategies that increase the probability of tunneling and thereby accelerate H* atom abstraction reaction rates.     

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Rate constants for the first two chemical steps of eumelanogenesis

The kinetics of the initial cyclization and redox exchange reactions involved in the eumelanogenic pathway have been studied previously but because of the difficulty of detecting the intermediate cyclodopa by optical means (because its absorbance is in the same range as dopa which is present in excess in the experimental system) no accurate value for the redox exchange reaction has so far been obtained and there is no available analytical methodology that can be applied to the successive first- and second-order reactions involved. We have synthesized cyclodopa and examined the kinetics of the formation of dopachrome following the pulse radiolytic generation of dopaquinone in its presence. From this direct measurement we determined that the rate constant of the reaction between cyclodopa and dopaquinone is 5.3 x 10(6)/M/s. Employing this value in a computational model of the combined cyclization and redox exchange reactions we calculate that the observed kinetics of dopaquinone decay and dopachrome formation are compatible with a cyclization rate constant of 3.8/s.