bluuebunny/crossref_metadata_2025 huggingface dataset:
SOLID‐PHASE PEPTIDE SYNTHESIS OF SOMATOSTATIN USING MILD BASE CLEAVAGE OF Nα‐9‐ FLUORENYLMETHYLOXYCARBONYLAMINO ACIDS
Authors: CHANG CHI‐DEU , FELIX ARTHUR M. , JIMENEZ MANUEL H. , MEIENHOFER JOHANNES
Published: August 2010

Experimental details for the “Fmoc solid phase peptide synthesis” of somatostatin are described. The 9‐fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor‐bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.‐butyl side chain protecting groups. Nα‐9‐fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di‐S‐protected somatostatin 14‐peptide was obtained in 55–60% overall yield. The S‐protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91% yield). Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.
PREPARATION OF A TWO‐DISULFIDE BONDED ENZYMICALLY ACTIVE DERIVATIVE FROM HEN EGG LYSOZYME
Authors: ACHARYA A. SEETHARAMA , TANIUCHI HIROSHI
Published: August 2010

A method has been developed for preparation of an enzymically active two‐disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23° for 40 min. The products are reacted with [1‐14C] iodoacetic acid and then purified by gel filtration and ion‐exchange chromatography. An enzymically active derivative containing 4 mol of [1‐14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1‐14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.
PROTEIN CONFORMATION WHEELS: CYTOCHROMES AND LYSOZYMES
Authors: SRINIVASAN A.R. , OLSON W.K.
Published: August 2010

Conformation wheels, directly relating the amino acid sequence to the local torsion angles in a protein molecule, are presented for cytochromes c, c2, c550, and c551 and for lysozymes from hen egg‐white and T4 bacteriophage. The circular plots for the cytochrome molecules aid in visualizing the common three‐dimensional folding (“cytochrome fold”) observed in this family of proteins. Conformation wheels for lysozymes from two different species reveal the characteristic differences in their folding patterns. These novel plots are also useful in storing and comparing the several sets of crystallographic data reported for lysozyme.
PREPARATION OF PROTEIN CONJUGATES WITH ALKOXYPOLYETHYLENE GLYCOLS
Authors: KING TE PIAO , WEINER COREY
Published: August 2010

Alkoxypolyethylene glycols can be converted to O‐alkoxypolyethyleneglycoxy‐S‐carboxamidomethyldithiocarbonates. These dithiocarbonates are useful for the preparation of protein conjugates with alkoxypolyethylene glycols. Studies with model compounds suggest that sites of reaction are primarily the amino groups of proteins and, to a lesser extent, the aliphatic and phenolic hydroxyl groups. Antigen E, which is an allergenic protein from ragweed pollen, was modified with different alkoxypolyethylene glycols by the above procedure. The size and the hydrophobicity of alkoxypolyethylene glycols used were found to influence the reduction in antigenic activity of conjugates.
COMPARISON OF STEREOSPECIFIC CONFORMATIONS FOR THREO AND ERYTHROβ‐HYDROXY‐N‐ACETYLTRYPTOPHANAMIDE
Authors: KARLE ISABELLA L.
Published: August 2010

Different isomers of β‐hydroxy‐N‐acetyl‐tryptophanamide are obtained depending upon the preparative route. The threo (from enzymatic oxidation) and erythro (from synthetic reduction) forms are distinguished further by their conformation, particularly by the severely restricted rotation about the Cβ‐Cγ bond, which appears to be independent of the presence of the hydroxyl group on Cβ. The torsional angle X21 about the Cβ‐Cγ bond is + 112° for the threo molecule and — 3° for the erythro molecule. Threo‐β‐hydroxy‐N‐acetyl‐L‐tryptophanamide (C13H15N3O3) crystallizes in space group P21 with cell parameters a = 13.572(4) Å, b= 6.296(1) Å, c = 13.796(4)Åand β= 148.10(2)°. Erythro‐β‐hydroxy‐N‐acetyl‐D L‐tryptophanamide (C13H15N3O3.1/2C6H6) crystallizes in space group P21/c with cell parameters a = 23.178(15) Å, b = 7.202(3)Å, c = 9.218(4)Åand β= 91.13(5)°.
DEGRADATION OF ENKEPHALIN AND ENKEPHALINAMIDE BY NEUROBLASTOMA x GLIOMA HYBRID CELLS AS STUDIED BY 13C N.M.R.
Authors: DESLAURIERS ROXANNE , JARRELL HAROLD C. , GRIFFITH DOUGLAS W. , MCGREGOR W. HERBERT , SMITH IAN C.P.
Published: August 2010

13C n.m.r. and thin‐layer chromatography were used to monitor the degradation of methionine‐enkephalinamide in the presence of neuroblastoma x glioma hybrid cells (NG 108‐15) and membranes. Puromycin and trypsin treatment failed to protect enkephalinamide from degradation over long periods of time (up to 24 hours). The major degradation products of [3[2‐13C]glycine] methionine‐enkephalinamide observed by 13C n.m.r. showed glycine‐3 in a non‐terminal position, an N‐terminal position and free glycine. A minor component showed glycine‐3 in a C‐terminal position.
PREPARATION AND SOME CHEMICAL CHARACTERISTICS OF MILK‐CLOTTING PROTEASE FROM BACILLUS MESENTERICUS 76
Authors: MESROB B.K. , VASSILEVA R.A.
Published: August 2010

Pure milk‐clotting protease (MCP‐76) is isolated by isotachophoresis at pH 5.0. The native molecule has only one protein chain. It is a metaloenzyme containing zinc. The pure MCP‐76 has a molecular weight of 33 000 (± 1500) and by diphenyl‐indenonyl‐isothiocyanate method showed arginine as N‐terminal amino acid.
PREPARATION AND CONFORMATIONAL STUDY OF CLUPEINE FRAGMENTS*
Authors: BONORA GIAN MARIA , BERTANZON FRANCESCO , TONIOLO CLAUDIO
Published: August 2010

Fragments of clupeines YI, YII, and Z of divergent chain length and different amino acid composition were prepared by digestion with thermolysin and a mixture of carboxypeptidases A and B, and their conformational preferences examined as a function of pH, added salts, presence of a helix‐supporting solvent, and temperature. All these highly basic oligopeptides adopt an essentially unordered conformation in aqueous solution. 2‐Chloroethanol supports in various amounts the onset of the right‐handed α‐helical form in the carboxypeptidase fragments.
β‐STRUCTURE OF POLYPEPTIDES IN NON‐AQUEOUS SOLUTIONS: I. Spectral Characteristics of the Polypeptide Backbone
Authors: RAGHAVENDRA K. , ANANTHANARAYANAN V.S.
Published: August 2010

The optical rotatory features of the β‐structure of the polypeptides in nonaqueous solutions and films cast from these solutions have been investigated. The β‐structure of poly‐S‐benzyl‐L‐cysteine, poly‐S‐carbobenzoxy‐L‐cysteine and poly‐S‐benzyl‐L‐cysteine, poly‐S‐carbobenzoxy‐L‐cysteine and poly‐O‐carbo‐bands of their films. The optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of these polypeptides are found to be very similar in both film and solution. In solvents promoting the β‐structure, the polypeptides are characterized by CD troughs in the n‐±* transition region of the peptide chromophore. The ORD spectra are found to be positive in sign throughout the visible and accessible ultraviolet regions and are interpreted in terms of the possible existence of a relatively much larger positive π‐π* CD band as compared with the negative π‐π* band. The rotatory data obtained in the non‐aqueous solution are compared with those obtained for other poly peptides in aqueous solutions, with respect to the type and extent of β‐structure present.
N‐METHYL PEPTIDES: III. Solution Conformational Study and Crystal Structure of N>‐Pivaloyl‐L‐Prolyl‐N‐Methyl‐N‘‐Isopropyl‐L‐Alaninamide*
Authors: VITOUX BERNARD , AUBRY ANDRE , CUNG MANH THONG , BOUSSARD GUY , MARRAUD MICHEL
Published: August 2010

The study of tBuCO‐l‐Pro‐Me‐l‐Ala‐NHiPr (1) by i.r. and n.m.r. spectroscopies has indicated that the middle amide group accommodates preferentially the cis arrangement in inert (CCl4) and aprotic (DMSO) solvents. Cis conformers are folded by a strong intramolecular hydrogen bond involving both terminal CO and NH groups whereas the minor trans conformers accommodate an open conformation. The cis folded form is retained in the solid state and its crystal structure was fully characterized by X‐ray diffraction.
DESIGN, SYNTHESIS AND 13 C‐ AND 1 H‐N.M.R. INVESTIGATION OF A CYCLIC OCTAPEPTIDE TO MIMIC THE ZINC‐BINDING SITE OF CARBOXYPEPTIDASE A
Authors: IYER K. SANKARANARAYANA , LAUSSAC JEAN‐PIERRE , LAU SHOW‐JY , SARKAR BIBUDHENDRA
Published: August 2010

Considering the three‐dimensional structure and the native Zn(II)‐binding ligands of carboxypeptidase A followed by extensive model building, a cyclic octapeptide, cyclo‐(Gly‐L‐Glu‐Gly‐Gly‐L‐His‐Gly‐L‐His‐Gly) was designed to mimic the Zn(II)‐binding site of carboxypeptidase A. The cyclic octapeptide was prepared by high dilution technique from the corresponding linear octapeptide, N‐Boc‐Gly‐γ‐OBut‐L‐Glu‐Gly‐Gly‐L‐His‐Gly‐L‐His‐Gly‐OBzlNO2 via the azide method. The linear octapeptide was obtained by coupling of the two tetrapeptide fragments: N‐Boc‐Gly‐γ‐OBut‐L‐Glu‐Gly‐Gly‐ONp and L‐His‐Gly‐L‐His‐Gly‐OBzlNO2. The cyclic peptide was purified to homogeneity by the method of countercurrent distribution. The product obtained was both ninhydrin negative and Pauli's reagent positive. Further confirmation of this material was obtained by the proper amino acid ratio of its acid hydrolysate and by the proton magnetic resonance spectrum in which the various kinds of protons of this peptide were accounted for. A detailed 13C‐ and 1H‐n.m.r. investigation was undertaken to determine the Zn(II)‐binding ligands of the cyclo‐octapeptide. The assignments for all the resonances were attempted by pH titration, by employing homonuclear decoupling experiments and by synthesis of cyclo‐octapeptide containing specifically deuterated amino acids cyclo‐(Gly‐L‐Glu‐Gly‐d2‐Gly‐d2‐L‐His‐Gly‐L‐His‐Gly). Titration results of Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)‐binding, the proton resonances were shifted downfield, the largest change being that of the histidine residues and more particularly C(2)‐H protons. The chemical shifts induced on glutamic acid residue were also observed for Glu CH2γ. In the case of 13C resonances, the maximum change in chemical shift was observed in the histidine residues and especially in the imidazole ring upon complexation. Two methylenes of the glutamic acid residue showed a large change in chemical shift upon ligating to the metal ion. The most significant observation was the deshielding effect of the Glu COO‐ group. The results demonstrate that Zn(II) binds both the imidazoles of the two histidine residues and the carboxyl side chain of the glutamic acid residue of the designed cyclic octapeptide.
SYNTHESIS OF SUBSTRATES SPECIFIC FOR HUMAN SPLEEN FIBRINOLYTIC PROTEINASE (SFP)
Authors: OKADA YOSHIO , TSUDA YUKO , NAGAMATSU YOKO , OKAMOTO UTAKO
Published: August 2010

Two peptide anilides, Suc‐Tyr‐Leu‐Val‐pNA (III) and Ac‐Ser‐His‐I.eu‐Val‐pNA (V) were synthesized by the conventional method with the object of obtaining specific substrates for human spleen fibrinolytic proteinase (SFP). The Kcat/Km values for hydrolysis of HI and V by SFP were 22 64 7 and 2 700, respectively. On the contrary, the Kcat/Km values for hydrolysis of III and V by porcine pancreatic elastase were 21 and 9, respectively.
COMPARATIVE STUDY OF THE HIGH MOLECULAR WEIGHT PROTEIN FRACTION OF MUSTARD (B. JUNCEA) AND RAPESEED (B. CAMPESTRIS)
Authors: RAO A. GURURAJ , RAO M.S. NARASINGA
Published: August 2010

A method is described for isolating the high mol. wt. protein fraction of mustard and rapeseed in a homogeneous form. The protein from the two species had nearly identical sedimentation coefficients, molecular weights, intrinsic viscosity and fluorescence emission spectrum. However, differences in the amino acid composition were observed. Data on circular dichroism and rate of hydrolysis by proteolytic enzymes suggested differences in the secondary structures and possibly amino acid sequence.
SYNTHESIS AND PROPERTIES OF HUMAN GROWTH HORMONE FRAGMENTS*
Authors: Pena Clara , Stewart J.M. , Ferrara P. , Paladini A.C.
Published: August 2010

The solid phase synthesis of fragments 75–120‐NH2 and 73–128‐GlyNH2 of the human growth hormone sequence is described. Purification was by solvent extraction, gel filtration and partition chromatography. No growth‐promoting activity was detected in any of the peptides by the rat body‐growth assay. An immunochemically reactive area was located in the sequence 73–128 when tested by complement fixation assays.
OVINE PROLACTIN: Isoelectricfocusing and Characterization of the Separated Components†
Authors: Cheng Christopher H.K. , Wong T.M. , Blake James , Li Choh Hao
Published: August 2010

Highly purified ovine prolactin was subjected to preparative isoelectricfocusing. Although complete separation of the isoelectrically different components was not achieved, four enriched fractions of these components were prepared by this method. Studies on the separated fractions revealed that these components of ovine prolactin, probably a result of deamidation processes, have rather different properties. The more acidic components appeared to be less active in both the radioreceptor assay on mammary gland membranes and the pigeon crop‐sac assay. However, the more acidic forms appeared to be more immunoreactive as revealed by radioimmunoassay.
CYCLIC PEPTIDES:
Authors: Noda Kosaku , Shibata Yukie , Gross Erhard , Shimohigashi Yasuyuki , Izumiya Nobuo
Published: August 2010

In order to establish possible routes for the synthesis of AM‐toxins, cyclotetradepsipeptides containing Dha, Pyr‐L‐Ala‐L‐Hmb‐L‐Tyr‐NH2 were prepared and treated with TFA or anhydrous HF. The product was identified to be cyclo (‐α‐Hyala‐L‐Ala‐L‐Hmb‐L‐Tyr‐), resulting from intramolecular condensation of the α‐keto and amide group with the formation of the α‐Hyala residue. Cyclo (‐α‐Hyala‐L‐Ala‐L‐Hmb‐L‐Phe‐) and cyclo(‐α‐Hyala‐L‐Ala‐L‐Val‐L‐Phe‐) were obtained by acid‐treatment of the corresponding pyruvyl‐tripeptide amides. The cyclic tetrapeptides did not show the activity of AM‐toxin.
Synthesis of N‐tert.‐butoxycarbonyl(α‐phenyl)aminomethylphenoxyacetic acid for use as a handle in solid‐phase synthesis of peptide α‐carboxamides
Authors: Gaehde Stephan A. , Matsueda Gary R.
Published: August 2010

N‐tert.‐butoxycarbonyl‐aminomethyl(α‐phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid‐phase synthesis of peptide α‐carboxamides. This handle was prepared with an 82% yield when N‐tert.‐butoxycarbonyl‐(p‐hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C‐terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH2Cl2 (< 1% loss after 10h). Upon treatment for 30min with HF:anisole(9:1) at 0°, 92% cleavage of glycinamide from Glyhandle‐resin was obtained. In a test synthesis of a peptide α‐carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.‐butoxycarbonyl‐aminomethylphenoxyacetic acid and N‐tert.‐butoxycarbonyl‐aminornethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid‐phase synthesis.
Enhancement of peptide coupling reactions by 4‐dimethylaminopyridine
Authors: Wang S.S. , Tam J.P. , Wang B.S.H. , Merripield R.B.
Published: August 2010

4‐Dimethylaminopyridine (DMAP) was found to be useful in the enhancement of peptide coupling reactions mediated by dicyclohexylcarbodiimide or symmetrical anhydrides. In an automated synthesis of the model heptapeptide Boc‐Ala‐Cle‐Ile‐Val‐Pro‐Arg(Tos)‐Gly‐OCH2‐Resin (Cle, cycloleucine), the efficiencies of various coupling methods such as dicyclohexylcarbodiimide, dicyclohexylcarbodiimide plus 1‐hydroxybenzotriazole, and symmetrical anhydride were compared with that of dicyclohexylcarbodiimide plus 4‐dimethylaminopyridine. Based on the amino acid composition of the peptide‐resin samples and high pressure liquid chromatographic analyses of the protected heptapeptide amide obtained from the ammonolytic cleavage of the peptideresin samples, it was concluded that only dicyclohexylcarbodiimide plus 4‐dimethylaminopyridine gave the desired near quantitative couplings in those cycles involving the sterically hindered amino acid residues. Observations were also made that 4‐dimethylaminopyridine was a useful additive in a modified symmetrical anhydride method of coupling. In the synthesis of the model tetrapeptide Leu‐Ala‐Gly‐Val on a Pam resin, the anhydride couplings were accelerated by DMAP and the product was equivalent in homogeneity to that obtained by the best previous methods. In addition, no racemization was detectable by a sensitive chromatographic method. There also was no detectable racemization found in a DCC‐DMAP coupling of Boc‐Ile‐OH with H‐Val‐OCH2‐resin. However, significant racemization was observed during the coupling of Boc‐Phe‐OH with H‐Glu(OBzl)‐OCH2‐resin. DMAP is recommended as an additive for coupling hindered amino acids, particularly Cα‐substituted residues, where little or no racemization is expected.
MOLECULAR DESIGN OF A CYCLIC HEPTAPEPTIDE TO MIMIC THE ZINC‐BINDING SITE OF CARBONIC ANHYDRASE.
Authors: Iyer K. Sankaranarayana , Laussac Jean‐Pierre , Sarkkar BIBUDHENDRA
Published: August 2010

A cyclic heptapeptide [cyclo‐(Gly‐L‐His‐Gly‐L‐His‐Gly‐L‐His‐Gly)] was designed to mimic the Zn(II)‐binding site of carbonic anhydrase. The cyclic heptapeptide was synthesized from the linear heptapeptide, Gly‐L‐His‐Gly‐L‐His‐Gly‐L‐His‐ Gly‐OH, which in turn was obtained by coupling of the fragments, viz. BOC‐Gly‐L‐His‐Gly‐N3 and L‐His‐Gly‐L‐His‐Gly‐OBzlNO2 followed by deblocking of amino and carboxyl protecting groups. Conversion of the linear heptapeptide to the azide by treatment with diphenylphosphoryl azide was followed by cyclization in high dilution. A homogeneous material was isolated by counter‐current distribution followed by gel filtration. It was found to be ninhydrin negative. The n.m.r. spectrum of the material upon integration indicated the proper ratios of various kinds of protons to be expected of the cyclic heptapeptide. A detailed 13C‐ and 1H‐n.m.r. investigation was undertaken to determine the Zn(II)‐binding ligands of the cyclic heptapeptide. The assignments for all the resonances were attempted by spin‐decoupling method, pH and solvent effects, and by comparison of resonances of similar protons and carbons of model peptides. The n.m.r. titration results of the Zn(II) bound form of the cyclic peptide showed the presence of a 1:1 complex. Upon Zn(II)‐binding, the changes in the chemical shift of the imidazole protons were relatively large, indicating that this ring is involved in the complexation. All the peptide ‐NH‐ resonances were observable and unaffected; consequently, none of these nitrogens can serve as a ligand. In the case of 13C resonances, addition of 1 equiv. of Zn(II) to the cyclic heptapeptide, the C(2), C(4), and C(5) carbon resonances of this group were dramatically affected and showed a very large change in chemical shift upon complexation. The results demonstrate that Zn(II) binds to all three imidazole residues of the designed cyclic heptapeptide.
Calorimetric study of tryptophan synthase α‐subunit and two mutant proteins
Authors: YUTANI KATSUHIDE , KHECHINASHVILI NIKOLAI N. , LAPSHINA ELENA A. , PRIVALOV PETER L. , SUGINO YOSHINOBU
Published: August 2010

Heat‐denaturation of tryptophan synthase α‐subunit from E. coli and two mutant proteins (Glu 49 ± Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20°, Cp20, has been found to be (0.43 ± 0.02) cal ± K‐1 ± g‐1, the unfolding heat capacity change, ΔdCp, (0.10 ± 0.01) cal ± K‐1 ± g‐1, and the unfolding enthalpy value extrapolated to 110°, Δdh110, (9.3 ± 0.5) cal ± g‐1 for the three proteins. The value of Cp20 was larger than those found for fully compact protein and that of Δdh110 was smaller. Unfolding Gibbs energy, ΔdG at 25° for Wild‐type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal ± mol‐1 at pH 9.3, respectively. Unfolding enthalpy, ΔdH, of the three proteins seemed to be the same and equal to (23.2 ± 1.2) kcal ± mol‐1 at 25°. As a consequence of the same value of ΔdH and the different value in ΔdG, substantial differences in unfolding entropy, ΔdS, were found for the three proteins. The values of ΔdG for the three proteins at 25° coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.
Preparation of photoreactive derivatives of α‐melanotropin by selective modification of the lysine or tryptophan residue
Authors: MURAMOTO KOJI , BUCKLEY DOUGLAS I. , RAMACHANDRAN J.
Published: August 2010

Photoreactive derivatives of α‐melanotropin were prepared by selective modification of the single tryptophan residue in position 9 or the amino group of the lysine residue in position 11 by reaction with 2‐nitro‐4‐azidophenylsulfenyl chloride. [2‐Nitro‐4‐azidophenylsulfenyl‐Trp9]‐α‐melanotropin was found to be five times as potent as α‐melanotropin in stimulating lipolysis in isolated rabbit adipocytes. [Nε‐2‐nitro‐4‐azidophenylsulfenyl‐Lys11]‐α‐melanotropin had only 11% of the lipolytic potency of α‐melanotropin.
Coupling in the absence of tertiary amines
Authors: BODANSZKY MIKLOS , BEDNAREK MARIA A. , BODANSZKY AGNES
Published: August 2010

In order to avoid base catalyzed side reactions during coupling, attempts were made to render superfluous the addition of tertiary amines to the reaction mixture. Weak acids were applied for the removal of acid labile protecting groups. Acetic acid and other carboxylic acids were considered unsuitable for this purpose because the resulting salts could be the cause of chain termination in the next coupling step. Pentachlorophenol and 2,4‐dinitrophenol cleaved the Bpoc, Nps and Trt groups but more practical rates were reached with solutions of 1‐hydroxybenzotriazole (HOBt) in trifluoroethanol, in acetic acid, or in a mixture of phenol and p‐cresol. In addition to acidolysis, HOBt salts of amino components could also be obtained through hydrogenolysis of the Z group or thiolysis of the Nps group in the presence of HOBt, or by the displacement of acetic acid from acetate salts with HOBt. Acylation of HOBt salts of amino components with symmetrical or mixed anhydrides or with active esters did not require the addition of tertiary amine.
Partial characterization of a subtilisin inhibitor from black bean seeds
Authors: SEIDL DINAH S. , ABREU HUGO , JAFFE WERNER G.
Published: August 2010

The subtilisin inhibitor (SI), isolated from black beans (Phaseolus vagaris) has a molecular weight of 8918 and it contains four half cystine residues. The N‐terminal residue is arginine and the C‐terminal is lysine. The subtilisin inhibiting site is composed of an Ala‐Leu or Ile bond. This site interacts in addition to subtilisin (S) with elastase from human leukocytes (HLE), both of which compete for the inhibitor. Also, HLE‐cleaved SI (SIHLE) is inactive against HLE as well as against S. It was shown that the inhibition of S by SI resembles a temporary inhibition, involving a very slow release of enzyme activity. The trypsin reactive site is a Lys‐Val bond, and its interaction with bovine trypsin is very weak. No complexes of SI or trypsin‐cleaved SI (SIT) with trypsin could be detected on cellulose‐acetate membrane electrophoresis, while SIT forms a clearly visible complex with S.
Conformations of cyclo(L‐alanyl‐L‐alanyl‐ε‐aminocaproyl) and of cyclo(L‐alany1‐D‐alanyl‐ε‐aminocaproyl); cyclized dipeptide models for specific types of β‐bends
Authors: BANDEKAR J. , EVANS D. J. , KRIMM S. , LEACH S. J. , LEE S. , McQUIE J. R. , MINASIAN E. , NEMETHY G. , POTTLE M. S. , SCHERAGA H. A. , STIMSON E. R. , WOODY R. W.
Published: August 2010

Conformational energy calculations indicate that the peptide backbones of the low‐energy conformations of the cyclized dipeptide derivatives cyclo (L‐alanyl‐L‐alanyl‐ε‐aminocaproyl) and cyclo (L‐alanyl‐D‐alanyl‐ε‐aminocaproyl) are constrained to form β‐bends of types I + III and II, respectively. Thus, the two compounds can serve as models for the spectroscopic properties of β‐bends of these types. The coupling constants obtained from 1H n.m.r. spectra in DMSO‐d6 are consistent with the dihedral angles of the computed lowest‐energy conformations. Differences in 13C chemical shifts between the two compounds can be correlated with differences in shielding by C=O groups in bends of various types. 1H and 13C chemical shifts suggest association of cyclo (L‐Ala‐L‐Ala‐Aca) but not of cyclo (L‐Ala‐D‐Ala‐Aca) in dimethylsulfoxide. The different tendencies to associate can be explained in terms of the difference in conformation. The circular dichroism spectra of the two compounds are quite different. In methanol, trifluoroethanol and water, the L‐Ala‐L‐Ala derivative has a positive extremum near 190 nm and two negative extrema near 206 and 220 nm, whereas the L‐Ala‐D‐Ala derivative has a positive extremum at about 203 nm and negative extrema at about 187 and 229 nm. The spectra can be used to estimate the contribution of various bend types in a related series of compounds. A normal mode analysis of the vibrations of the computed low‐energy conformations was compared with solid state infrared and Raman spectra, in order to determine the predominant conformations. The bend types determined by this comparison fully agree with the predictions of the theoretical computations for both derivatives.
Conformation and structure of acidic dipeptides
Authors: EGGLESTON DRAKE S. , HODGSON DEREK J.
Published: August 2010

The crystal structure of the acidic dipeptide glycyl‐L‐aspartic acid dihydrate, Gly‐L‐Asp·2H2O, C6H10N2O5·2H2O, has been determined by means of three‐dimensional counter X‐ray data. The dipeptide crystallizes in space group P21 21 21 of the orthorhombic system with four formula units in a cell of dimensions of a= 9.611(4), b= 9.775(6), and c= 10.845(4) Å. The structure was solved by direct methods and refined by least‐squares methods to a final value of the weighted R‐factor of 0.035 using all 1191 observed intensity data. The dipeptide occurs in the crystal as a zwitterion, with the glycyl N‐terminus protonated and the aspartyl main chain carboxyl group deprotonated. The conformation about the peptide bond is fully trans extended, with an ω angle of—175.9°. While there is extensive intermolecular hydrogen bonding in the crystals, there is no intramolecular hydrogen bonding either here or in the solid state structures of related dipeptides.
β‐Endorphin
Authors: YAMASHIRO DONALD , LI CHOH HAO , NICOLAS PIERRE , HAMMONDS R. GLENN
Published: August 2010

A double‐headed analog of human β‐endorphin (βh‐EP),N, N'‐bis(β‐endorphinyl)‐cystine (II), has been synthesized by the solid‐phase method, along with βh‐EP‐Cys(CH2CONH2)‐OH (I) and (Tyr31]‐βh‐EP (III). Their relative potencies in a radioreceptor‐binding assay were: Bh‐EP, 100; II, 235; I, 170; and III, 204. In the tail‐flick test for analgesic activity their relative potencies were: βhEP, 100; II, 86; I, 93; and III, 116.
Crystal and molecular structure of tert.‐butyloxycarbonyl‐L‐hydroxy‐prolyl‐α‐aminoisobutyryl‐α‐aminoisobutyryl‐L‐phenylalaninol
Authors: VAN ROEY PATRICK , SMITH G. DAVID , BALASUBRAMANIAN T.M. , REDLINSKI A.S. , MARSHALL GARLAND R.
Published: August 2010

The crystal structure of the synthetic tetrapeptide, Boc‐Hyp‐Aib‐Aib‐Phol, an analogue of the C‐terminal tetrapeptide in the antibiotic Antiamoebin I, was determined as part of a study of the conformation of peptaibophol antibiotics. The crystals are orthorhombic, space group P212121, with cell parameters a = 16.576 (1) Å, b= 17.657 (1) Å, c = 10.435 (1) Å, V = 3053.9 (2) Å3, Z = 4, Dc= 1.163 g · cm‐3. The three amino acids form a single turn of a 310‐helix, stabilized by two intramolecular hydrogen bonds. The Aib residues adopt the usual conformation in the region between the 310‐ and α‐helices. The terminal hydroxy methyl group of the phenylalaninol residue is disordered. The position of the benzyl side chain of the amino alcohol relative to the backbone corresponds to a conformation also observed in phenylalanine residues.
Unusual cis‐trans isomerism in N‐acetyl, N‘‐methylamide derivatives of syn‐ and anti‐5‐methylproline
Authors: DELANEY NORMA G. , MADISON VINCENT
Published: August 2010

By means of carbon‐13 n.m.r. spectroscopy, we have investigated cis‐trans isomerism for N‐acetyl, N'‐methylamide derivatives of syn‐ and anti‐5‐methylproline (syn: the methyl group and carboxamide are on the same side of the proline ring, anti: on opposite sides). For Ac‐syn‐5‐MeProNHMe, we observe about 25%cis peptide bond isomers in most solvents as is common for oligopeptides of proline. The percentage of cis isomer is about 20% greater for Ac‐anti‐5‐MeProNHMe in non‐polar solvents. These percentages show little temperature dependence. The free energy barrier for trans to cis isomerization is 19.4 kcal/mol in dioxane and 20.0 kcal/mol in dimethylsulfoxide for Ac‐anti‐5‐MeProNHMe, in agreement with values observed for other peptides. However, values observed for Ac‐syn‐5‐MeProNHMe are about 2 kcal/mol lower. For the latter peptide the barrier is 1.7 kcal/mol higher in water than in three organic solvents. These results indicate that steric interactions of the 5‐methyl group destabilize the trans peptide isomer in Ac‐anti‐5‐MeProNHMe without affecting the isomerization barrier. In contrast, for Ac‐syn‐5‐MeProNHMe, the cis and trans peptide isomers are destabilized by the same amount so that the barrier is lowered without altering the equilibrium ratio. The higher energy barrier in water indicates binding of solvent to the planar peptide ground state.
Circular dichroism studies of relaxin and insulin peptide chains
Authors: DU YU‐CANG , MINASIAN ELIZABETH , TREGEAR G.W. , LEACH S.J.
Published: August 2010

The circular dichroism (CD) spectra of pig relaxin and pig insulin are similar, reflecting the known structural similarities between the two hormones. However, the conformational characteristics of the separate chains of insulin and relaxin show significant differences. The S‐sulfo forms of insulin A and B chains and S‐sulfo relaxin A chain have CD spectra consistent with largely unordered structures whereas the S‐sulfo form of relaxin B‐chain has at least 90%β‐structure. This β‐structure may explain the unusual solubility and adsorptive properties of the relaxin B‐chain and the poor combination yields with A‐chain. The relaxin B‐chain changes to a largely unordered conformation if the peptide is shortened at the carboxyl terminus by six amino acid residues. This conformational change has important implications in planning relaxin synthesis strategy. Significant interactions and conformational changes are observed between the oxidized forms of the A and B chains of both relaxin and insulin. In using CD to monitor chain recombination of native relaxin peptides it was found that the spectra obtained differ depending on whether the reduced chains are separated or not separated from the reaction mixture prior to reoxidation. Although the spectra differ the biological activity was 20% in both cases. The remainder of the reoxidized but inactive material contains β‐structures which make a greater contribution to the CD spectrum when the chains have been separated and processed than when they are reoxidized in situ.
Studies on the disulfide region of α1‐protease inhibitor
Authors: GLASER CHARLES B. , KARIC LUCIJA , HUFFAKER TIM , CHANG RUTH , MARTIN JOHN
Published: August 2010

The single disulfide bond of purified human α1‐protease inhibitor was reduced with dithiothreitol in the absence of denaturant and the resultant sulfhydryl groups were alkylated with iodoacetamide‐1‐C14. The product was found to be fully functional as an inhibitor of trypsin and elastase in esterolytic and proteolytic assays. The modified protein was also found to be nearly identical to native α1‐protease inhibitor when analyzed by immunological, electrophoretic, and spectral methods. The performic acid oxidized inhibitor, on the other hand, was devoid of any enzyme inhibitory activity. Analysis of the derivatized protein by amino acid analysis and by radioactive counting revealed only a single cysteine‐containing peptide. The alkylated inhibitor was digested with cyanogen bromide and then trypsin, and subjected to two‐dimensional peptide mapping. A single cysteine‐containing peptide was recovered and shown to have the sequence Phe‐Asn‐Ile‐Gln‐His‐Cys‐Lys. A variety of experiments involving gel filtration or dialysis of reduced or oxidized α1‐protease inhibitor indicate that this Cys‐peptide is covalently bound to either free cysteine or to glutathione via a disulfide bridge.
Peroxide‐induced conformational transitions of peanut storage protein (arachin)
Authors: JACKS T.J. , HENSARLING T.P. , MULLER L.L. , ANGELO A.J. ST. , NEUCERE N.J.
Published: August 2010

Several effects of peroxides on protein have been described in the literature. Some of the effects have been ascribed to alterations in protein conformation but this has never been examined directly. This paper describes responses of peanut storage globulin (arachin) to rancid oil and hydrogen peroxide as measured by electrophoretic mobility, antigenicity and circular dichroism of the globulin. Rancid oil and 3% hydrogen peroxide had little effect on these properties but 30% hydrogen peroxide increased the mobility, abolished the antigenicity and reduced the ordered structural modes of the protein. The results indicated that changes in protein conformation do not occur after contact with organoleptically rancid oil or dilute peroxides but do occur with concentrated peroxides.
Human β‐endorphin
Authors: CABASSI FRANCO , ZETTA LUCIA
Published: August 2010

1H spectra at 270MHz of the βh‐endorphin glycyl residues in aqueous solution are reported. The chemical shifts, coupling constants and temperature coefficients are compared with those of the glycyl residues in Met‐enkephalin and in a random coil model peptide. The local conformation of Tyr‐Gly‐Gly‐Phe‐segment observed in Met‐enkephalin is maintained in βh‐endorphin.
Solid‐phase synthesis of chicken vasoactive intestinal peptide by a mild procedure using Nα‐9‐fluorenylmethyloxycarbonyl amino acids
Authors: COLOMBO ROBERTO
Published: August 2010

The octacosapeptide amide corresponding to the entire amino acid sequence of chicken vasoactive peptide (VIP) was assembled on a p‐benzyloxybenzylamine resin support using the base‐labile 9‐fluorenylmethyloxycarbonyl as Nα‐protecting group, cleaved by mild acid treatment, and purified by gel‐filtration and ion‐exchange chromatography. The symmetrical anhydride coupling was employed and monitored by two independent methods, and acetic anhydride termination was incorporated to minimize formation of deletion peptides. The homogeneity of the final product, obtained in 18% yield, was assessed by t.l.c., disc electrophoresis, amino‐terminal amino acid analysis, and amino acid analyses of acid and enzyme hydrolysates. The purified chicken VIP was shown to be active on gastric acid secretion and on pancreatic blood flow. Previously reported ring closure of the Asp‐Asn unit seemed to be at a minimum, owing to the mild basic and acid treatments.
Protection of methionine in peptides during iodination by sulfonium salt formation
Authors: BIENERT MICHAEL , KLAUSCHENZ ERHARD , NIKOLICS KÁROLY , NIEDRICH HARTMUT
Published: August 2010

A method for the prevention of methionine oxidation during iodination of tyrosine containing peptides is reported. The methionine containing peptide is converted into the corresponding S‐tert.‐butylsulfonium derivative, which is iodinated using iodine monochloride. After removal of the S‐tert.‐butyl group and purification, sulfoxide‐free 3,5 diiodotyrosine (Dit) peptides were obtained. Dit8‐substance P, Dit8‐physalaemin6–11 and Dit1, Met5‐enkephalin were synthesized by this route. Tritium labeling of Dit1, Met5‐enkephalin yielded 3H‐enkephalin with a specific radioactivity of 38 Ci/mmol.
Synthesis of low‐phenylalanine polypeptides
Authors: Nakhost Z. , Hsieh D.S.T. , Shih V. , Rha C.K.
Published: August 2010

Low‐phenylalanine‐peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by α‐chymotrypsin then followed by carboxypeptidase‐A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight > 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soybean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.
Crystal and molecular structure of tert. ‐butyloxycarbonyl‐L‐prolyl‐α‐aminoisobutyryl‐L‐alanyl‐α‐aminoisobutyrate methyl ester
Authors: van ROEY PATRICK , SMITH G. DAVID , BALASUBRAMANIAN T.M. , CZERWINSKI E.W. , MARSHALL GARLAND R. , MATHEWS F. SCOTT
Published: August 2010

Boc‐Pro‐Aib‐Ala‐Aib‐OMe crystallizes in the orthorhombic space group P21212 with cell dimensions a = 17.701 (3)Å, b = 17.476 (4)Å, c = 9.686 (2)Å, V = 2996.3 Å3. The first three residues form a single turn of a 310‐helix stabilized by two intramolecular hydrogen bonds. Comparison of the conformation of the methyl ester of the tetrapeptide with that of its benzyl ester shows differences in the individual torsion angles of up to 29°, although the overall conformation is conserved.
Synthesis and biological activity of ΔPhe4‐enkephalins*
Authors: SHIMOHIGASHI YASUYUKI , STAMMER CHARLES H. , COSTA TOMMASO , VONVOIGTLANDER PHILIP F.
Published: August 2010

The ΔPhe4‐enkephalins have been synthesized and examined in an in vitro receptor binding assay and an in vivo tail flick analgesia test. The ΔPhe4 residue was derived from Boc‐Gly‐Phe(β‐OH)‐OH by spontaneous dehydration and azlactonization. The dipeptide azlactone was coupled directly with H‐Leu‐OBzl to yield a tripeptide which was converted into the pentapeptides after stepwise coupling with two amino acids using the water soluble EDC‐HOBt method. Dehydroenkephalins were liberated with hydrogen fluoride in the presence of anisole. In the radioligand binding assay which did not contain an enzyme inhibitor [d‐Ala2, ΔPhe4, Leu5] enkephalin was almost twice as active as saturated [d‐Ala2, d‐Leu5] ‐enkephalin. The ΔPhe4‐enkephalins exhibited a considerably diminished activity as compared with the saturated peptide in the in vivo analgesic assay. These results are discussed with regard to the enzyme stability and receptor preference of dehydroenkephalins.
Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance
Authors: Parsons Thomas F. , Pierce John G.
Published: August 2010

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C‐2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (α or β) have had their accessible histidines exchanged with deuterium, permitted assignment of C‐2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine α‐83; in isolated α subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit‐subunit interaction.
Use of synthetic gramicidins in the determination of channel structure and mechanism
Authors: URRY D.W. , TRAPANE T.L. , ROMANOWSKI S. , BRADLEY R.J. , PRASAD K.U.
Published: August 2010

The syntheses of (1‐13C) Trp9 gramicidin A (GA), (1‐13C) Trp11 GA, (1‐13C) Trp13 GA, (1‐13C) Trp15 GA, and D · Leu2 GA are verified by means of high performance liquid chromatography, carbon‐13 nuclear magnetic resonance, circular dichroism and characterization of transport properties. The use of these and other synthetic gramicidins is discussed in terms of determining ion binding sites within the channel, helix sense of the channel, the basis of monovalent vs divalent cation selectivity, and means of modulating channel conductance.
Minimum information content and formation of interacting ribonuclease fragment complexes
Authors: KANMERA TATSUHIKO , HOMANDBERG GENE A. , KOMORIYA AKIRA , CHAIKEN IRWIN M.
Published: August 2010

The degree to which amino acid sequence can be simplified with retention of conformational and functional properties has been investigated by semisynthesis using non‐covalent fragment complexes of bovine pancreatic ribonuclease as test cases. Based on the ribonuclease S system, a set of synthetic model sequences was defined for the S‐peptide (1–20) region which interacted productively with native S‐protein (21–124). The most simple sequence, an eicosapeptide containing helix‐favoring Ala residues at all positions except Glu 1 and 14, Phe 8, His 12, and Met 13, effected at least 15% of ribonuclease catalytic activity (versus native ribonuclease S) when added to S‐protein in saturating amounts. The data for model S‐peptides define an α‐helical framework and specific side chains at positions 8, 12, and 13 as the core of sequence information necessary for S‐peptide to effect a productive non‐covalent complex with S‐protein. Previous ribonuclease fragment studies also were used as a basis for making the productive, non‐overlapping complex, (1–15):(21–111):(116–124). Addition of synthetic (1–15) and (116–124) to (21–111) led to a 3d̀ increase in Tm and 4% (versus ribonuclease A) catalytic activity. The three‐fragment complex, with the β‐bend residues 112–115 deleted, exhibited significantly lower stability to thermal denaturation than did related two‐fragment complexes. The potential use of three‐fragment complexes related to the above is discussed for semisynthetic sequence modeling concomitantly in the N‐ and C‐terminal regions of ribonuclease.
4‐Chloromethylphenoxyacetyl polystyrene and polyamide supports for solid‐phase peptide synthesis
Authors: COLOMBO ROBERTO , ATHERTON ERIC , SHEPPARD ROBERT C. , WOOLLEY VIVIENNE
Published: August 2010

Two functionalised supports for the solid‐phase synthesis of peptides under mild reaction conditions were prepared: 4‐chloromethylphenoxyacetamidomethylcopoly (styrene‐1%‐divinylbenzene) and 4‐chloromethylphenoxyacetyl‐norleucylpoly(dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base‐catalyzed esterification of the first protected amino acid to the 4‐alkoxybenzyl alcohol resins formerly employed in combination with Nα‐9‐fluorenylmethoxycarbonyl and tert.‐butyl side‐chain protecting groups. Esterification of Nα‐protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4‐chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu‐enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion‐exchange chromatography and reverse phase high pressure liquid chromatography.
Synthesis and biological activity of glycosylated analogs of the C‐terminal hexapeptide and heptapeptide of Substance P
Authors: POUJADE C. , LAVIELLE S. , TORRENS Y. , MARQUET A.
Published: August 2010

The synthesis of two glycosylated analogs of Substance P is described. The activity of the peptides was assayed on the isolated guinea‐pig ileum and their degradation was studied using rat hypothalamus slices. While glycosylation noticeably enhances the solubility of the corresponding compounds, the β‐glucopyranosyl moiety only slightly modifies the biological half‐life and the bioactivity of the glycopeptides.
Stabilization of β‐turn conformations in enkephalins
Authors: SUDHA T.S. , BALARAM P.
Published: August 2010

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α‐aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly.1H n.m.r. studies of Tyr‐Aib‐Gly‐Phe‐Met‐NH2, Tyr‐Aib‐Aib‐Phe‐Met‐NH2and Tyr‐Gly‐Aib‐Phe‐Met‐NH2demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t‐butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β‐turn centred at positions 2 and 3 is proposed for the Aib2‐Gly3analog. In the Gly2‐Aib3analog, the β‐turn has Aib3‐Phe4as the corner residues. The Aib2‐Aib3analog adopts a consecutive β‐turn or 310helical conformation. Highin vivobiological activity is observed for the Aib2and Aib2‐Aib3analogs, while the Aib3peptide is significantly less active.
Peptides containing dipropylglycine
Authors: HARDY PAUL M. , LINGHAM IAN N.
Published: August 2010

Using 4‐heptanone in the Ugi reaction, it is possible to prepare a peptide containing a single residue of dipropylglycine (Dpg) in modest yield, but attempts to form peptides containing contiguous Dpg residues were unsuccessful. Methods of extending Dpg‐Dpg to higher homo‐oligomers have been examined. Carboxyl extension of N‐trifluoroacetyl (Tfa)‐Dpg2 is possible through its oxazolin‐5(4H)‐one, but only as far as the tripeptide. However, amino extension of Dpg2‐OButby successive steps of addition of 2‐trifluoromethyl‐4,4‐dipropyloxazolin‐5 (4H)‐one and N‐deprotection allowed preparation of Tfa‐Dpg6‐OButin good yield. Removal of the Tfa group from Dpg residues is only possible using sodium borohydride reduction under conditions which lead to partial reduction of the t‐butyl esters of protein amino acids. The use of the N'N'‐dibenzylhydrazide (DBH) group for C‐protection, however, circumvents this problem. Direct regeneration of carboxyl from DBH is possible with bromine in acetonitrile, and catalytic reduction gives the free hydrazide. Tfa‐Dpg‐NHNH2 can be oxidatively coupled to Gly‐OButbut not to Dpg‐OBut. Tfa‐Dpg3‐N3 undergoes Curtius rearrangement in preference to peptide bond formation, and Dpg3‐N3 eliminates isocyanic acid on heating to form N‐(Dpg)2‐heptylideneimine.