3-Thiopropionic acid as a highly versatile multidetachable thioester resin linker

This paper describes a practical new use of3-mercaptopropionic acid as a highly versatilemultidetachable linker for solid-phase synthesis. Ourapproach is based on the stability of thealkylthioester functionality to optimized Boc-SPPSprotocols and HF treatment, as well as on the mildactivation of the thioester functionality towardnucleophilic or reductive displacement. This allowsseveral C-terminal modifications to be introduced intoa synthetic molecule during the cleavage step. We haveshown that unprotected peptides can be efficientlycleaved from a propyl thioester-polyethyleneglycol-poly-(N,N-dimethylacrylamide) copolymerresin using a great variety of nucleophiles to givethe corresponding C-terminally modified peptides(esters, thioesters, carboxylic acids, thioacids,amides, hydroxamic acids, hydrazides, alcohols). Thenucleophilic cleavage reaction is both rapid andexceptionally clean in all the cases tested.     

Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible post-translational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce solid-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh₃)₄. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of ¹H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by ¹H-tetrazole and followed by oxidation led to the methylphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

3-(1-Piperidinyl)alanine formation during the preparation ofC-terminal cysteine peptides with the Fmoc/t-Bu strategy

Summary Several side reactions have been detected for cysteine-containing peptides. During the synthesis ofC-terminal cysteine peptides, a base-catalyzed elimination of the sulfhydryl-protected side-chain to afford the dehydroalanine derivative followed by a nucleophilic addition to the alkene was observed. MALDI-TOF analysis was a useful analytical technique to determine this phenomenon.Abbreviations Acm acetamidomethyl - Boc tert-butyloxycarbonyl - t-Bu tert-butyl - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - Fmoc 9-fluorenylmethyloxycarbonyl - HATU N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HBTU N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HOAt 1-hydroxy-7-azabenzotriazole - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - PAC 4-hydroxymethylphenoxyacetic acid handle - PAL 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxy-phenoxy)valeric acid handle - PEG-PS polyethylene glycol-polystyrene graft supports - PS polystyrene - Reagent R TFA/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2) - S-t-Bu S-tert-butyl mercapto - TFA trifluoroacetic acid - Trt triphenylmethyl. Amino acid symbols denote thel-configuration, unless indicated otherwise     

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N′-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N′-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl₂·2H₂O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid–phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid).     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Purification of the c-erbB2/neu membrane-spanning segment:: a hydrophobic challenge

High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography–ESI TOF mass spectrometry.     

Allylic protection of thiols and cysteine. III. Use of Fmoc-Cys(Fsam)-OH for solid-phase peptide synthesis

Summary The solid-phase synthesis of peptides containing Cys has been carried out using the new thiol protecting group Fsam, which is completely stable to basic and acidic conditions used in both main strategies and can be selectively removed by palladium-catalyzed allylic cleavage in the presence of nucleophiles. This protecting group adds a new dimension of orthogonality for regioselective cysteine pairing strategies. Abbreviations: Those used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature inJ. Biol. Chem. 1972,247, 977–983. The following additional abbreviations are used: Acm,S-acetamidomethyl; Alloc, allyloxycarbonyl; BME, β-mercaptoethanol; Boc,tert-butyloxycarbonyl; Bu, butyl;tBu,tert-butyl; Bzl, benzyl; DIEA,N,N-diisopropylethylamine; DIP-CDI,N,N′-diisopropylcarbodiimide; DMF,N,N-dimethylformamide; EtOAc, ethyl acetate; Fm, fluorenylmethyl; Fmoc, fluorenylmethyloxycarbonyl; Fmoc-AM,p-[(R,S)-α-[1-(9HFluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid; Fnam,S-[N-[2,3,5,6-tetrafluoro-4-(N′-piperidino)-phenyl],N-allyloxycarbonyl]-aminomethyl Fsam,S-[N-[2,3,5,6-tetrafluoro-4-(phenylthio)-phenyl],N-allyloxycarbonyl]-aminomethyl; HOAc, acetic acid; HOBt, 1-hydroxybenzotriazole; Meb,S-4-methylbenzyl; Mmt,S-4-methoxytriphenylmethyl; Mob,S-4-methoxybenzyl; NDMBA,N,N′-dimethylbarbituric acid; Npys,S-3-nitro-2-pyridinesulfenyl; OPac, phenacyl ester; PEG-PS, poly(ethyleneglycol)-polystyrene graft resin support; Ph, phenyl; Phacm,S-phenylacetamidomethyl; StBu,S-tert-butylmercapto; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tmob,S-2,4,6-trimethoxybenzyl; Trt,S-triphenylmethyl. Amino acid symbols denote the L-configuration unless indicated otherwise.     

Using cyclic peptide mixtures as probes for metal ion host-guest interactions

Summary Mixtures of cyclic peptides, formed by head-to-tail cyclizations of side-chain resin-bound linear sequences, have been prepared using solid-phase synthesis. Fast atom bombardment mass spectrometry of cyclic peptides with various metal ions can reveal preferred modes of host-guest patterns, albeit in a nonquantitative manner. This approach could prove useful for more rapid screening of potential peptide ionophores. A cyclic heptapeptide with a dipeptide tail proved to be a particularly effective host for a Ca²⁺ ion; in a small three-component mixture, cyclo[Gly-Asp-d-Pro-Xxx-Asp-d-Pro-Asp(Aca-Phe-NH₂)], binding to Ca²⁺ varied from Xxx=N-MeAla>GlySar. In a 15-component mixture, cyclo[Pro-Xxx-Asn-Pro-Xxx-Asn] where Xxx=Ala, Glu, Leu, Lys or Phe, there were no significant differences with respect to binding to metal ions. We believe this to be the first reported use of cyclic peptide libraries for screening metal ions to discern host-guest relationships.Abbreviations Aca aminocaproic acid - Boc tert-butyloxycarbonyl - BOP benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate - DCM dichloromethane - DIEA diisopropylethylamine - DMF N,N-dimethylformamide - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - pMBHA 4-methylbenzhydrylamine - TFA trifluoroacetic acid This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

A method for the facile solid-phase synthesis of gramicidin S and its analogs

Summary A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete ¹H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.Abbreviations Boc t-butyloxycarbonyl - BOP benzotriazoyl N-oxytris(dimethylamino)phosphonium hexafluorophosphate - Bzl benzyl - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DQF-COSY double-quantum-filtered correlation spectroscopy - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate - HOBt 1-hydroxybenzotriazole - 4-MeBzl 4-methylbenzyl - NHS N-hydroxysuccinimide - NOESY nuclear Overhauser effect spectroscopy - PAM phenylacetamidomethyl (resin) - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoroacetic acid - TOCSY total correlation spectroscopy - Tos tosyl - Troc 2,2,2-trichloroethylcarbamate.     

Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Summary Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent,N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphateN-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

Evaluation of coupling conditions for the incorporation of Nα-Fmoc-Tyr(PO3H2)-OH in solid-phase peptide synthesis

Summary In order to minimise the formation of the pyrophosphate derivative of the target peptide when side-chain-unprotected phopshotyrosine is used in solid-phase peptide synthesis, this building block can be incorporated using benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole/N-methylmorpholine (1:1:2.3) in the presence of a chaotropic salt (0.4 M LiCl in N-methyl-2-pyrrolidinone).Abbreviations BOP benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate - DIEA diisopropylethylamine - Fmoc 9-fluorenylmethoxycarbony - HATU N-[(dimethylamino)1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethan-aminium hexafluorophosphate N-oxide - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser-desorption ionization time-of-flight mass spectrometry - NMM N-methylmorpholine - NMP N-methyl-2-pyrrolidinone - Pmc 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl - ® solid support - TFA trifluoroacetic acid - TPTU 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyluroniumfluoroborate. Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]     

Sequence determination of peptides by mass spectrometry: methods for combining "wet" separation techniques with mass spectrometry.

Methods are described that allow the combination of established techniques for peptide separation, paper chromatography and electrophoresis, with mass spectrometry. The development of these methods is part of an ongoing effort in the search for a methodology for the systematic utilization of mass spectrometry for the elucidation of primary structure of proteins and peptides. Peptides and amino acids are detected on chromatograms by conversion to covalent derivatives that are also suitable for mass spectrometry. The most useful reagents for detection and derization of peptides reported here are dansyl chloride, N,N-dimethylaminobenzaldehyde, N,N-dimethylaminocinnamaldehyde, and N-hydroxysuccinimido β-naphthoate. Detection limits and mass spectra for some of these derivatives are reported.     

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with ¹⁵N, ¹⁵N/¹³C and even ¹⁵N/¹³C/²H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.     

I. Photoaffinity probes provide a general method to prepare synthetic peptide-conjugates

A general synthetic strategy is described for the preparation of peptide-conjugates where the peptides contain the NH₂ terminal, COOH terminal, or internal regions of the protein sequence. Glycoprotein D of herpes simplex virus type 1 is used as a representative protein. Ten-residue peptide fragments of the native sequence were synthesized using standard solid-phase methodology. Photoprobes stable to conditions of synthesis and HF cleavage were coupled directly to the protected-peptide resin during synthesis. This one-step procedure eliminates the potential modification of functional groups in the sequence of interest that can occur when using chemically labile bifunctional reagents. Since the photoprobe is inert until photolysis, the synthetic peptide-probe can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The following photoprobe derivatives were investigated: thep-azidobenzoyl,p-nitrophenylalanyl, andp-benzoylbenzoyl groups. The benzophenone photoprobes were shown to give the highest incorporation of peptide-probe with the protein carrier over a wide range ofpH and solvent conditions. For solid-phase synthesis three benzophenone photoprobes can be used: benzoylbenzoic acid, benzoylbenzoylglycine, andN ^e-(4-benzoylbenzoyl)-N -t-butyloxycarbonyl-lysine.     

Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy

Summary In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.This work was taken in part from the Ph.D. Theses of E.A. Ottinger (1994) and Q. Xu (1996), University of Minnesota, Minneapolis, MN, U.S.A. Preliminary presentations of portions of this work were made at the Twenty-Second European Peptide Symposium, Interlaken, Switzerland, September 13–19, 1992, see Ref. 1, at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995, and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995, see Ref. 2. The title side reaction was first discussed for tyrosine (see Refs 1 and 3), but all of the mechanism studies discussed herein are for serine and threonine.Amino acid symbols denote the l-configuration, and abbreviations for amino acids and peptides follow rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977].     

Solution phase synthesis of the 14-residue peptaibol antibiotic trichovirin I

Summary.  The 14-residue peptaibol antibiotic trichovirin I 4A of the structure Ac-Aib-L-Asn-L-Leu-Aib-L-Pro-L-Ala-L-Val-Aib-L-Pro-Aib-L-Leu-Aib-L-Pro-L-Leuol (Aib = α-aminoisobutyric acid, Leuol = leucinol) was synthesized by stepwise conventional solution phase synthesis using the Z/OtBu(OMe) strategy and HOBt/EDC as coupling reagents. Intermediates were fully characterized and the identity of the synthetic peptide with the component 4A of the natural, microheterogeneous peptide mixture was proven by electrospray mass spectrometry, HPLC, and bioassay. Received March 25, 2002 Accepted June 14, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated to Prof. Dr. Günther Jung. Tübingen University, on the occasion of his 65^th anniversary. Authors' address: Prof. Dr. Hans Brückner, Interdisciplinary Research Center, Institute of Nutritional Science, Department of Food Sciences, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany, Fax: +49-641-99-39149, E-mail: hans.brueckner@ernaehrung.uni-giessen.de Abbreviations: Amino acids are abbreviated according to three-letter-nomenclature; Aib, α-aminoisobutyric acid (2-methylalanine); Iva (isovaline, 2-ethylalanine); Leuol, L-leucinol [(S)-2-amino-4-methyl-1-pentanol]; AAA, amino acid analysis; EI-MS, electron impact mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; Z, benzyloxycarbonyl; Fmoc, 9-fluorenylmethyoxycarbonyl; OtBu, tertiary butoxy (tert-butylester); OMe, methoxy (methyl ester); OBzl, benzyloxy (benzyl ester); TDM, N,N,N′,N′-tetramethyl-4,4′-diamino-diphenylmethane (Arnold's base); for other abbreviations see Experimental.     

Betaine lipids in chlorarachniophytes

Evolving from the endosymbiosis of a green algal cell by a filose amoeba or amoeboflagellate, the chimearic chlorarachniophytes combine unique features retained from both of their ancestral units. They have preserved from the endosymbiont only the nucleomorph and chloroplast. Four strains from three genera of this algal class were studied to identify a set of non‐phosphorous‐containing polar lipids and their associated fatty acids using the techniques of positive‐ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Fourteen non‐phosphorous‐containing polar lipids, classified as betaine lipids were primarily identified as forms of diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) and its structural isomer diaclyglycerylhydroxymethyl‐N,N,N‐trimethyl‐β‐alanine (DGTA). Though the number of forms of DGTA and DGTA were roughly equal, DGTS composed more of the polar lipid portion present in three of the strains tested, while the fourth, Lotharella globosa, was dominated by forms of DGTA. In addition, a lipid tentatively identified as diacylglycerylcarboxyhydroxymethylcholine (DGCC) was observed twice in minor amounts. The polar lipid‐associated fatty acids of the aforementioned algal strains generally included dodecanoic acid (12:0), tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), and eicosapentaenoic acid [20:5(n‐3)]. The differences in betaine lipid content among the species studied may allow for further conclusions to be drawn regarding the taxonomy of chlorarachniophytes.     

Synthesis of N-protected peptides by the o-nitrophenylsulfenyl group using o-nitrophenylsulfenyl N-carboxy α-amino acid anhydrides

N-protected peptides, which are important intermediates as a carboxyl component in the fragment condensation method, have been prepared in high yields by the reaction of o-nitrophenylsulfenyl (Nps) N-carboxy α-amino acid anhydrides with unprotected peptides and amino acids in aqueous organic systems. An Nps hexapeptide ester was prepared by the fragment condensation of an Nps tripeptide with a tripeptide ester. It was demonstrated that the synthesis of unprotected peptides by the NCA method, followed by N-protection by the Nps-NCA, is a rapid and very useful method for preparing Nps peptides.     

Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

p‐Methoxyphenol: A potent and effective scavenger for solid‐phase peptide synthesis

During the final step of t‐Boc/Bzl, solid‐phase peptide synthesis (SPPS)‐protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support . These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during S_N1‐type reactions. Five peptide sequences were synthesised for evaluating p‐methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin–peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p‐cresol (reported in the literature as an effective acyl ion eliminator) and p‐methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p‐methoxyphenol can be used as a potent scavenger during SPPS by t‐Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.