The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide.

Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.     

The case of sulfonation in the chemical synthesis of oligonucleotides

The sulfonation of nucleosidic component, a side reaction during phosphotriester bond formation, as a function of the reactivity of the condensing agents and the kind of substituents in the starting phosphodiester is discussed. It was found that in the coupling reaction of nucleoside alkyl phosphodiesters, the degree of sulfonation of the nucleosidic component was very high; while under the same conditions when the aryl group was present in the corresponding phosphodiester, this side reaction practically did not occur.     

Mechanistic studies on the phosphoramidite coupling reaction in oligonucleotide synthesis. I. Evidence for nucleophilic catalysis by tetrazole and rate variations with the phosphorus substituents.

Tetrazole catalyzed reactions of a series of phosphoramidites, 5'-O-DMTdT-3'-O-P(OR1)NR2(2) (1a-h), with 3'-O-SiButPh2-6-N-benzoyl-dA (2a) in acetonitrile solution have been studied. It is found that the coupling rate depends very much on whether tetrazole is added before or after 2a, and that dialkylammonium tetrazolide salts are inhibitors. These and other facts are evidence that the reactions are subjected to nucleophilic catalysis by tetrazole, in addition to acid catalysis. The rate variations with phosphorus substituents of 1a-h are NEt2 greater than NPri2 greater than N(CH2CH2)O greater than NMePh, and OMe greater than OCH2CH2CN greater than OCHMeCH2CN greater than OCMe2CH2CN much greater than OC6H4Cl. The inhibitor properties of dialkylammonium tetrazolides have practical consequences for the efficiency of DNA syntheses, when in situ prepared phosphoramidites are used; the same would apply for segmented, simultaneous syntheses or syntheses where recycling is performed.     

Synthesis of cyclopentapeptides and cycloheptapeptides by DEPBT and the influence of some factors on cyclization.

Three cyclic peptides - cyclo(GlyAlaTyrLeuAla), cyclo(GlyProTyrLeuAla) and cyclo(GlyTyrGlyGlyProPhePro) - isolated and identified from medicinal herbs were chosen as model cyclic peptides to study the influence of the linear precursors and coupling reagents on cyclization. The 17 linear precursors of these three cyclic peptides were synthesized and cyclized using 3-(diethoxyphosphoryloxy)-(1-3)-benzotriazin-4 (3H)-one (DEPBT) as the major coupling reagent. The present work shows that: (i) the effects of linear peptide precursors on the cyclization are complex but some guidelines for choosing suitable precursor for cyclization could be considered; and (ii) DEPBT results in a higher cyclization yield compared with other coupling reagents. In addition, it was confirmed that peptides containing alternating D and L residues favor cyclization.     

Sequence-Dependent Structural Variations of DNA Revealed by Chemical Ligation

Abstract

The comparative study of nick-sealing efficiency under the action of BrCN or water-soluble carbodiimide was carried out for 14 dinucleotide combinations in double helix. The difference between the lowest (17%, GpG) and the highest (94%, CpT) coupling yields was found to be more than five fold, both condensing reagents showing a similar sequence-specific trend. A strong correlation observed between coupling yields at different dinucleotide combinations and ³¹P NMR parameters supports the idea that variations in chemical ligation efficiency arise from sequence-specific modulations of the helix geometry and confirms close similarity of the intrinsic fine structure of intact and nicked DNAs.     

Syntheses of Terpenes by the Condensation of Aliphatic Compounds

The condensation of mesityloxide with 4-diethylaminobutanone-(2) in the presence of potassium t-butoxide gave the products containing piperitenone together with isoxylitones, which were identified by gas chromatography and chemical methods. The effects of the combination of condensing agent, solvent, molar ratio of reagents and reaction temperature on the components in the condensation products were studied. It was found that piperitenone was able to obtain in an yield of 51% on the basis of 4-diethylaminobutanone-(2) by the use of Triton-B as a condensing agent. Also piperitenone can be isolated from the condensation products by the treatment with semicarbazide.     

Simplications in the synthesis of short oligonucleotide blocks.

A rapid and convenient procedure has been developed for the synthesis of fully protected mono, di and trideoxyribonucleotides utilizing an aryl phosphoroditriazolide. It affords advantages over coupling strategies employing condensing reagents, such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide in preparing small oligonucleotides and is relatively free of the drawbacks inherent in other approaches using bifunctional phosphorylating reagents. In particular, the synthesis of trinucleotide blocks without purification at the dimer stage is described.     

The 'azirine/oxazolone method' in peptaibol synthesis: preparation of a derivative of trichotoxin A-50 (G)

The synthesis of a mixture of epimeric derivatives of the peptaibol trichotoxin A-50 (G) is described. The 'azirine/oxazolone method' has been used as a superior method for the introduction of the Aib as well as the Iva units into the peptide chain. In this protocol, 2,2-disubstituted 2H-azirin-3-amines are the synthons for 2,2-disubstituted glycines, which undergo coupling with N-protected amino or peptide acids in high yield, and without any need of coupling reagents. The problem of the instability of the amide function of the Gln side chain under the conditions of the acid-catalyzed hydrolysis of Z-Gln-(Aib)(n)-N(Me)Ph was solved by using an appropriate protecting group for the amide function of the Gln side chain, e.g., the triphenylmethyl (trityl; Tr) group. The structures of two intermediate peptides, i.e., the segments comprising residues 1-5 and 10-13, resp., were established by X-ray crystallography.     

Template-directed polymerization of oligoadenylates using cyanogen bromide

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.     

Studies on the role of tetrazole in the activation of phosphoramidites.

The mechanism of the tetrazole-activated coupling step in the synthesis of oligonucleotides via phosphoramidites is studied with the help of model reactions: Treatment of diethoxydiisopropylaminophosphane with two equivalents of tetrazole resulted in a diethoxy-tetrazolophosphane, whose (31P)-NMR shift of 126 ppm is identical with the signal observed during internucleotide bond formation. A series of different related diethoxy-phosphorous-acid derivatives were also synthesized; their (31P)-NMR signals between 123.9 and 130.8 ppm are additional evidence for the intermediacy of a tetrazolide species. Further NMR investigations with more basic azoles showed that tetrazole is also active as a proton donor.     

Cyclization studies with tetra- and pentapeptide sequences corresponding to beta-casomorphins

The tetrapeptide Boc-D-Orn-Phe-D-Pro-Gly-OH and the pentapeptide sequence Boc-Tyr(tBu)-D-Orn-Phe-D-Pro-Gly-OH were used to study the influence of different coupling reagents on the yield and purity of these model peptides. The simple structure prevented racemization and cyclodimerization and facilitated the ring formation. The most favorable effects on yield and purity were obtained in both reactions using diphenyl-phosphoryl azide (DPPA) and norborn-5-ene-2,3-dicarboximidodiphenylphosphate (NDPP), while the cyclizations with the powerful activating reagents benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with the exception of the cyclopentapeptide reaction with HBTU/4-dimethylaminopyridine gave unsatisfactory results.     

Triazine-based condensing reagents

The synthesis, properties, and application of condensing reagents derived from 1,3,5-triazines are described. The mechanism of activation of carboxylic function, structure of reactive intermediates, and mechanism of acylation of nucleophiles are presented. The synthetic versatility of mono- and bifunctional reagents for syntheses in solution, triazine-based immobilized reagents, chiral triazines for enantiodifferentiating syntheses, are discussed. The scope and limitation of the synthetic utility of triazine reagents in the preparation of heterocyclic compounds, amides, esters, oligopeptides-including large-scale syntheses and use in the combinatorial chemistry-is demonstrated.     

Discovery of new photoactivatable diaryltetrazoles for photoclick chemistry via 'scaffold hopping'

We report the discovery of two long-wavelength (365 nm) photoactivatable diaryltetrazoles through screening a small library of diaryltetrazoles that were designed using a ‘scaffold hopping’ strategy. A naphthalene-derived tetrazole showed excellent reactivity in the photoinduced cycloaddition reaction with methyl methacrylate under 365 nm photoirradiation in acetonitrile PBS buffer mixture. Besides, the brightly fluorescent pyrazoline cycloadducts that were formed further increase the potential utility of these new diaryltetrazoles as ‘photoclick’ reagents and as reporters in biological studies.     

Studies on the Synthesis of Oligonucleotides via the Hydrogenphosphonate Approach

Abstract

³¹P NMR studies on the activation and coupling of nucleoside 3-hydrogenphosphonates with nucleosides in the presence of various condensing agents, are discussed.     

Kinetics of azide attack on organonitriles coordinated to pentaamminecobalt(III)

Four new 5-substituted tetrazole complexes of pentaamminecobalt(III) have been synthesized. The N-1 bonded complexes of 5-(p-methylphenyl)tetrazole, 5-(p-chlorophenyl)tetrazole, 5-(p-nitrophenyl)tetrazole and the N-2 bonded complex of 5-(m-formylphenyl)tetrazole. Kinetic studies of the complex formation reaction, wherein azide ion attacks coordinated organonitrile ligands, have been shown to follow a second order rate law, first order in nitrile complex and azide. Rate constants and activation parameters have been determined for five organonitrile complexes and a Hammett type correlation has been established for the formation reaction described by the equation log k = 2.42σ – 2.44 at 25 °C and ionic strength 1.0 M. Two mechanisms are proposed for complex formation which are consistent with our observations.     

Enzymatic semisynthesis of [LeuB30] insulin

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.     

Synthesis of symmetric and unsymmetric 1,4-bis(p-R-phenylethynyl)benzenes via palladium/copper catalyzed cross-coupling and comments on the coupling of aryl halides with terminal alkynes

A series of symmetric 1, 4-bis(p-R-phenylethynyl)benzenes (6a-h) have been prepared via Pd¹¹/Cu¹ catalyzed cross- coupling of 1, 4-diiodobenzene (5) and p-substituted phenylethynes (4a-h). Similarly, the unsymmetric analogues (9a-c) were obtained from 1-iodo-4-(p-nitrophenylethynyl)benzene (8) and p-substituted phenylethynes (4c, 4d, 4g). Quantitative analysis of 1,4-(trimethylsilyl)butadiyne (10), produced in the catalytic coupling of ethynyltri- methylsilane with aryl halides using PdCl₂(PPh₃)₂/CuI in an amine solvent, confirmed that catalyst initiation proceeds via reduction of Pd¹¹ to Pd⁰ with concomitant oxidative homo-coupling of two ethynyltrimethylsilane molecules producing exactly one equivalent of 10 based on Pd¹¹. If air is present, the PdCl₂(PPh₃)₂/CuI/amine mixture provides a very effective system for catalytic oxidative homo-coupling of terminal alkynes to diynes and thus air must be rigorously excluded from the cross-coupling reactions. Hydrodehalogenation can compete effectively with the cross-coupling reaction for highly fluorinated aryl halides. Under certain conditions, the fluorinated aryl bromide or iodide can serve as the oxidant for the alkyne to diyne oxidative homo-coupling reaction. This can be avoided by appropriate choice of reaction conditions and reagents. These competing pathways have significant implications for the cross-coupling of aryl halides with terminal alkynes and are discussed herein.     

A new sensitive Edman-type reagent: 4-(N-1-dimethylaminonaphthalene-5-sulfonylamino)phenyl isothiocyanate. Its synthesis and application for micro-sequencing of polypeptides

A new fluorescent PITC homologue Edman-type reagent, 4-(N-1-dimethylaminonaphthalene-5-sulfonylamino)phenyl isothiocyanate (DNSAPITC), was synthesized following a simple three-step synthetic route. The reagent was crystallized and characterized by thin-layer chromatography, IR and electron impact mass spectrometry. Reference DNSAPTH-amino acid derivatives were prepared and a two-dimensional chromatography system on micro-polyamide sheets was developed for separating this mixture. On these sheets the sensitivity was 1-5 pmol, by exposure at 366 nm. Model peptides and proteins were subjected to Edman degradations with this new reagent. A similar coupling efficiency and repetitive degradation yield to those of PITC were found with this reagent. The advantages and limitations of this reagent for sensitive microsequencing are discussed.     

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt‐based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine‐assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6‐trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′‐dimethoxydiphenylmethyl and 4‐methoxybenzyloxymethyl groups were demonstrated to be acid‐labile S‐protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave‐assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.     

Easy parallel screening of reagent stability,quality control,and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1‐{[1‐(Cyano‐2‐ethoxy‐2‐oxo‐ethylidenaminooxy)dimethylamino‐morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma‐Aldrich, St. Louis, MO, USA) by SPPS of Leu‐Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3‐aminopropyl)triethyoxysilane‐modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.