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

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

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.     

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

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 hexafluorophosphate N-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.     

Convenient solid phase synthesis method for the preparation of cysteine C-terminally derivatized peptides

Summary This paper describes a novel solid phase peptide synthesis method for the systematic C-terminal modification of cysteine-containing peptides. In this method, cysteine is linked to chloromethylated polystyrene resin by its thiol functionality, followed by protection of the N-terminus and derivatization of the carboxylic acid to esters or amides. We report here on examples of the methodology and its application to the synthesis of Ac-Asp-cyclo(Cys-Gly-Pro-Cys)-NHBzl, a cyclic peptide amide. The method has been applied to the synthesis of complex esters as well as amides.Abbreviations Ac acetyl - AcN acetonitrile - Ac₂O acetic anhydride - AcOH acetic acid - Boc t-butyloxycarbonyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - cHex cyclohexyl - DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA diisopropylethylamine - DMF dimethylformamide - DMS dimethylsulfide - HOB 1-hydroxybenzotriazole - MBzl 4-methyl benzyl - MeOH methanol - TEA triethylamine - TEAP triethylammonium phosphate - TFA trifluoroacetic acid     

BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.     

Synthesis of a biotinylated, iodinatable, and photoactivatable probe for angiotensin receptors

We propose here a biotinyl-aminohexanoyl-[Ala1, Phe(4N3)8]angiotensin II analog as a radioiodinatable and photoactivatable probe for covalent labeling, detection and isolation of angiotensin receptors. A combination of solid phase and minimum-protection segment-coupling strategy using hexafluorophosphate of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium (BOP) as a coupling reagent is proposed for the synthesis of this probe. Optimized yields were obtained by HPLC monitoring of all reactions. A complete n.m.r. study suggests an extended conformation of this molecule, allowing a simultaneous recognition of receptor and avidin. The probe binds with high affinity (Kd = 2 nM) to angiotensin II receptors from rat liver membranes.     

Chemical synthesis and NMR characterization of structured polyunsaturated triacylglycerols

The chemical synthesis of pure triacylglycerol (TAG) regioisomers, that contain long chain polyunsaturated fatty acids, such as arachidonic acid (AA) or docosahexaenoic acid (DHA), and saturated fatty acids, such as lauric acid (La) or palmitic acid (P), at defined positions, is described. A single step methodology using (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), an activator of carboxyl group commonly used in peptide synthesis and occasionally used in carboxylic acid esterification, has been developed for structured TAG synthesis. Identification of the fatty acyl chains for each TAG species was confirmed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and fatty acid positional distribution was determined by (1)H and (13)C NMR spectra. The generic described procedures can be applied to a large variety of substrates and was used for the production of specific triacylglycerols of defined molecular structures, with high regioisomeric purity. Combination of MS and NMR was shown to be an efficient tool for structural analysis of TAG. In particular, some NMR signals were demonstrated to be regioisomer specific, allowing rapid positional analysis of LC-PUFA containing TAG.     

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.     

Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides.

This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH(2), and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DMF; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH(2)Cl(2)/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine), 2,3,5,6-tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6-di-tert-butyl-4-(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

Chemical synthesis of 15N-labeled zervamicins IIB]

Analogues of 16-membered peptide antibiotic zervamicin IIB with the Gln3 and Gln11 residues 15N-labeled at the C alpha-atoms were synthesized by coupling the antibiotic segments (1-4), (5-9), and (10-16). In turn, these were prepared by a stepwise chain elongation in solution starting from their C-termini using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as an activating agent. The sterically hindered 2-aminoisobutyric acid was introduced by the BOP-dimethylaminopyridine system with the preactivation of the carboxyl component. The segment condensation was performed with the use of the 6-trifluoromethylbenzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate activating reagent. The homogeneity of the resulting zervamicin analogues was confirmed by HPLC, and their structures were proved by NMR spectroscopy and FAB mass spectrometry.     

In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis

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

Novel Synthesis of <Emphasis Type="Italic">N</Emphasis>-Glycosyl Amino Acids Using T3P<Superscript>®</Superscript>: Propylphosphonic Acid Cyclic Anhydride as Coupling Reagent

In this paper is presented a novel and simple synthetic pathway for obtaining new protected and unprotected N-glucosyl amino acids from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl amine and Fmoc-l-amino acids. Three methodologies were evaluated, using the coupling reagents: N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate, diisopropylcarbodiimide and propylphosphonic acid cyclic anhydride. The obtained products using propylphosphonic acid cyclic anhydride showed less undesired species, easy purification and higher yields than the other two methodologies. Deprotection strategies widely used in solid phase peptide synthesis were applied to develop the synthetic pathway reported and achieve the final products. The protected and unprotected N-glucosyl amino acids were purified using solid phase extraction chromatography and characterized by high performance liquid Chromatography and nuclear magnetic resonance spectroscopy. Different amino acids (Fmoc-l-Asp(OtBu)OH, Fmoc-l-Phe(OH) and Fmoc-l-Lys(Boc)-OH) have been employed to demonstrate the simple and reproducible coupling methodology using propylphosphonic acid cyclic anhydride. The results showed that new protected and unprotected N-glucosyl amino acids can be obtained with high purity and the methodology could be used with any Fmoc-amino acid. The methodology developed could be considered as a synthetic tool for obtaining building blocks for glycopeptide synthesis and potential drugs candidates based on glycoconjugates.     

Chemical synthesis of human beta-endorphin(1-27) analogs by peptide segment coupling. Leucine and glycine residues bearing thiocarboxyl functions as junctions for peptide segment coupling.

[Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2, two analogs of human beta-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides. Msc-[Gly8]beta hEP(1-8)SH and Msc-[L-Leu8]beta hEP(1-8)SH, were synthesized by standard solid phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[alpha-(Boc-L-Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 in the segment coupling reaction were approximately 18%. Less than 1% of racemization of Leu-8 occurred during coupling of Msc-[L-Leu8]beta hEP(1-8)SH to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of beta-endorphin(1-27) and its analogs influenced the receptor binding activity.     

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Coupling of iminodiacetic acid with amino acid derivatives in solution and solid phase

Summary The IR studies for the preactivation step of N-protected iminodiacetic acid with different coupling reagents (TCFH, TFFH, HATU, HBTU, HSTU) were reported here and showed the formation of an anhydride as an active intermediate in case of TCFH and TFFH. The formation of a mixture of an anhydride and an active ester (-OBt,-OAt or-OSu) were observed for HBTU, HATU or HSTU coupling reagent. Dependent on the coupling conditions, acylation of N-protected iminodiacetic acid with amino acid ester or amide derivatives in solution phase gave monoor di-substituted iminodiacetic acid derivatives. Coupling of N-protected iminodiacetic acid with an amino acid or peptide attached to a solid support (PAL-PEG-PS or Wang resin) gave only the monosubstituted iminodiacetic acid derivatives. Abbreviations: HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; Boc,t-butyloxycarbonyl; DCC, N,N′-dicyclohexylcarbodiimide; DIC, N,N′-diisopropylcarbodiimide; DIEA, diisopropylethylamine; HATU, N-[(dimethylamino)-1H-1,2,3,-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; DMF, N,N-dimethylformamide; Bsmoc, 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl; Fmoc, 9-fluorenylmethyloxycarbonyl; HOAt, l-hydroxy-7-azabenzotriazole; HOBt, l-hydroxybenzotriazol; IDA, iminodiacetic acid; HSTU, O-(succinimidyl)-tetramethyluronium hexafluorophosphate; TCFH; 1,1,3,3-tetramethyl-2-chloroformamidinium hexafluorophosphate; TFFH, 1,1,3,3-tetramethyl-2-fluoroformamidinium hexafluorophosphate; TMS-Cl, trimethylchlorosilane. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem., 247 (1972) 997).     

Nα-Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride: A convenient building block for the solid-phase synthesis of phosphotyrosyl peptides

Summary Fmoc-O,O-(dimethylphospho)-l-tyrosine was converted into stable Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride by means of (diethylamino) sulfur trifluoride or cyanuric fluoride. This building block was used for efficient coupling of phosphotyrosine to the adjacent sterically hindered amino acid Aib or Ac₆c in, model peptide sequences as well as for the synthesis of the ‘difficult’ phosphotyrosine peptide Stat91^695–708. The phosphate methyl groups were cleaved on solid phase after peptide assembly by means of trimethylsilyl iodide in MeCN. Aib, α-aminoisobutyric acid Ac₆c, 1-amino-cyclohexyl-l-carboxylic acid; BOP, benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate, CIP, 2-chloro-l, 3-dimethylimidazolidium hexafluorophosphate, DAST, (diethylamino)sulfur trifluoride; DBU 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIEA, drisopropylethylamine; DMA dimethylacetamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,O-(7-azabenzotriazol-l-yl)-1.1,3,3-tetramethyluronium hexafluorophosphate; HOAt, I-hydroxy-7-azabenzotriazole; HOBt,N-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; MBHA, 4-methylbenzhydrylamine; MeCN, acetonitrile; NMP,N-methyl-2-pyrrolidinone; NMR, nuerear magnetic resonance; PS, polystyrene; PyBroP, bromotris(pyrrolidino)phosphonium hexafluorophosphate; Rink amide MBHA-PS, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminophenyl)-phenoxyacetamido-norleucyl-MBHA-PS; TFA, trifluoroacetic acid; TMSI, trimethylsilyl iodide; TPTU, 2-(2-pyridon-l-yl)-1,1,3,3-tetramethyluroniumfluoroborate; t_R, retention time; UNCA, arethane-protected amino acidN-carboxy anhydride Abbreviations for amino acids and nomenclature of, peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9].     

A convenient incorporation of conformationally constrained 5,5-dimethylproline into the ribonuclease A 89-124 sequence by condensation of synthetic peptide fragments.

The presence of l-5,5-dimethylproline (dmP) within an amino acid sequence results in the formation of an X-dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C-terminal 36-residue peptide, corresponding to the 89-124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5- and 31-residue peptide fragments, which were synthesized by solid-phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-dmP-OH pentapeptide (residues 89-93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate was found to be superior to bromo-tris-pyrrolidino-phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the -Tyr(tBu)-dmP- peptide bond in solution as well as on a resin.     

Synthesis and secondary structural studies of penta(acetyl-Hmb)A beta(1-40).

The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native A beta(1-40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of A beta(1-40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)A beta(1-40) was found to adopt both alpha-helix and intermolecular beta-sheet conformations. In 100% TFE a mixed alpha-helix/random coil structure was formed by the protected peptide indicating reduced alpha-helical propensity relative to A beta(1-40). The protected peptide formed beta-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native A beta(1-40), penta(acetyl-Hmb)A beta(1-40) did not form large aggregate species.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80).