bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of transmembrane β‐strands from hydrophobic characteristics of proteins
Authors: GROMIHA M. MICHAEL , PONNUSWAMY P.K.
Published: August 2010

The assembly of outer‐membrane proteins consisting of β‐strands as transmembrane segments is somewhat more complex when compared to the assembly of inner membrane proteins having α‐helices as transmembrane parts. This is probably due to the difference in the amino acid sequences of the transmembrane part strands and helices. Because of this feature, most predictive schemes which are successful in predicting transmembrane helical segments fail to predict transmembrane strand segments. Here we propose a new predictive scheme for forecasting the transmembrane strand segments in outer‐membrane proteins with the use of the general surrounding hydrophobicity scale developed both for the globular and membrane proteins in the preceding article. Two major features of the scheme are (i) that it does not solely depend on the amphipathic character of a sequence segment while identifying it as a transmembrane strand, and it is capable of predicting strands in varied lengths, a facility to reflect the variation in the membrane surfaces. This scheme predicts the transmembrane β‐strands in porin from R. capsulutus at 76% accuracy (giving due weights to over‐ and under‐predictions when compared to X‐ray results). The predicted β‐structure contents in OmpA, porin from E. coli and maltoporin compared with the Raman spectroscopic results at 95% level. These accuracy levels are far superior than the levels obtained from other existing methods. Apart from the above four proteins for which experimental informations are available, ten other outer‐membrane proteins, for which there is no information about their secondary structure, are considered in the forecast. The results are analysed and the coninion features in the folds of the set of fourteen outer‐membrane proteins are deduced.
Influence of methylation of the histidine ring of [Sar1]angiotensin II on conformation and biological activity
Authors: MOORE GRAHAM J , GOGHARI MAHESH H , FRANKLIN KEVIN J
Published: August 2010

[Sar1His(1‐Me)6]ANG II and [Sar1His(3–Me)6]ANG II were synthesized by the solid‐phase method and purified by reversed‐phase HPLC. 1H‐NMR spectroscopy at 400 MHz demonstrated the presence of two major conformers for both peptides in DMSO, representing cis and trans isomers (ratio 1:3 and 1:4, respectively) due to restricted rotation at the His‐Pro bond. The contractile activities of these peptides in the rat isolated uterus assay were <0.1 and 27% of that of ANG II, respectively. The bioactivities of these analogues were mirrored in their NMR spectra: the inactive analogue [Sar1His(1‐Me)6]ANG II showed perturbations of the Sar and His residues which were not present for [Sar1His(3‐Me)6]ANG II and the reference agonist [Sar1] ANG II. The high activity of the analogue methylated at His N3 suggests that an ionizable imidazole proton is not an absolute requirement for expression of biological activity by angotensin ligands, and that the imidazole group in the molecule may function in an ion dipole‐based mechanism when an intramolecular proton transfer (charge relay) mechanism is not available. The biological activity of the ligand appears to depend on the degree of proton transfer from Tyr‐OH to the imidazole acceptor, wherein complete (formal) proton transfer represents 100%, activity.
Conformational investigation of α,β‐dehydropeptides
Authors: LISOWSKI MAREK , PIETRZYŃSKI GRZEGORZ , RZESZOTARSKA BARBARA
Published: August 2010

Conformations of three series of model peptides: homochiral Ac‐Pro‐L‐Xaa‐NHCH3 and heterochiral Ac‐Pro‐D‐Xaa‐NHCH3 (Xaa=Phe, Val, Leu. Abu. Ala) as ivell as α,β‐dehydro Ac‐Pro‐ΔXaa‐NHCHs [ΔXaa = (Z)‐ΔPhe, ΔVal. (Z)‐ΔLeu, (Z)‐ΔAbu] were investigated by CD spectroscopy in 2 % dichloromethanecyclohexane, trifluoroethanol. water. and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane‐cyclohexane and unordered ones occur in water. The folded conformers are of the inverse γ‐turn type for all the peptides but Ac‐Pro‐L‐Phe‐NHCH3 for which the type‐I β‐turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the typc‐II β–turn in 2% dichloromethanecyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2%, dichloromethanecyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the βII‐turn conformation as determined for these coinpounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.
The evaluation of type I and type II β‐turn mixtures
Authors: PERCZEL A. , HOLLOSI M. , SANDOR P. , FASMAN G.D.
Published: August 2010

Circular dichroism (CD) and1 H‐{1H}NOE spectra were obtained for Piv‐Pro‐Ser‐NHCH3(1),[Piv‐(CH3)3‐C‐CO], Boc‐Pro‐Ser‐NHCH3 (2) and Boc‐Val‐Ser‐NHCH3 (3), to determine the solution conformation of these p‐turn models. In the crystal, 1 and 3 adopt an ideal type I β‐turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc‐Pro tert‐amide bond. The predominance of a β‐turn conformation in solution was suggested for models 1‐3 on the basis of 1H‐{1H}NOE data. In a nonpolar solvent the prevailing trans rotamer form (>80%) of 2 has a β‐turn conformation according to heteronuclear NOE measurement. Positive 1H‐{1H} NOEs were detected between the Hα(Pro)/NH(Ser), Hα(Ser)/NH(Ser) and NH(NHCH33)/HN(Ser) protons in the trans Boc‐Pro rotamer form of 2 at ‐20° in CDCl3. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc‐Val‐Ser‐NHCH3 (3). Boc‐Val‐D‐Ser‐NHCH3 (4) and Boc‐Pro‐D‐Ser‐NHCH3 (5), in various solvents. The 1H‐ {1H)NOE experiments carried out in CD3CN clearly showed that besides the type I (or III) β‐turn structure, one of the main conformations of models 1‐5 is close to the type II β‐turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1‐3 were characterized by class C (helix‐like) CD spectra, although class C spectra are generally only correlated with the type I β‐turn conformation. These acyclic models are the first carefully investigated examples of ‐L‐L‐ triamide systems, containing a significant amount of a type II β‐turn, as well as the type I p‐turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were ‘calibrated’ using the 1H‐{1H}NOE data. Such a “calibration”, as well as the semi‐quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C’ CD spectra may be obtained from the linear combination of the same two‐component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.
Comparison of GH‐stimulation by GH‐RH(1‐29)NH2 and an agmatine29 GH‐RH analog, after intravenous, subcutaneous and intranasal administration and after pulmonary inhalation in rats
Authors: PINSKI JACEK , YANO TETSL , GROOT KATE , ZSIGO JOZSEF , REKASI ZOLTAN , COMARU‐SCHALLY ANA MARIA , SCHALLY ANDREW V.
Published: August 2010

Many studies have shown that human GH‐RH(1‐29)NH2 possesses full intrinsic activity of GH‐RH(1‐44)NH2in vitro and in vivo. This investigation was performed to evaluate the efficacy of GH‐RH(1‐29)NH2 given by different routes of administration in stimulating GH release in rats. In each case GH‐RH(1‐29)NH2 was administered intravenously, subcutaneously, intranasally and by pulmonary inhalation at two different doses to groups of seven male rats. At a dose of 150 μg/kg GH‐RH(1‐29)NH2, the magnitude of GH response was significantly higher for the pulmonary inhalation group (355 ± 33.2 ng GH/mL) than for the subcutaneous group (246 ± 36ng GH/mL) or for the intranasal group (175 ± 30ng GH/mL). The group injected intravenously with GH‐RH(1‐29)NH2 at a dose of 2.5 μg/kg showed the highest response, GH levels reaching 877.2 ± 115 ng/mL. A similar pattern of responses was obtained for the superactive GH‐RH(1‐29) agmatine29 analog, MZ–3‐149, at doses that were 50 times lower. Our results indicate a high bioavailability of GH‐RH(1‐29)NH2 or analog MZ‐3‐149 administered by a convenient pulmonary inhalation route. The GH‐releasing effect of GH‐RH(1‐29)NH2 or analog MZ‐3‐149 delivered by pulmonary inhalation is superior to subcutaneous and intranasal administration.
Serine‐containing 10‐membered cyclodepsipeptides
Authors: CERRINI S. , GAVUZZO E. , LUIS G. , PINNEN F.
Published: August 2010

As a part of a Research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N‐phenylacetyl‐seryl‐propyl‐proline tripeptides has been examined. Two 10‐ membered peptide lactones, and , have been isolated by treating the corresponding linear p‐nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X‐ray analysis. Both the 10‐membered rings of 5a and 5b adopt an overall cis‐cis‐trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from Δω= 30° for the DSer—Pro bond in 5b to Ao= 5‐6° for the DSer‐PrO bond in 5a and PrO—DPrO bond in 5b. The skeletal atoms of 5b, containing the PrO‐DPrO sequence, are related by a pseudo‐symmetry mirror plane passing through the Pro carbonyl and the opposite DSer CβH2 group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer‐Pro ring junction; this arrangement gives rise to a C5‐type ring structure which is well evidenced in the case of 5a.
Studies on the carbodiimide‐mediated model couplings of Z‐Pro‐Leu‐OH with benzoaza‐15‐crown‐5
Authors: KULIKOV NICOLAY V. , BASOK STEPHAN S. , LUKYANENKO NICOLAY G.
Published: August 2010

A series of model DCC‐mediated couplings of Z‐Pro‐D‐Leu‐OH with 2,3‐benzo‐10‐aza‐1,4,7,13‐tetraoxacyclopentadeca‐2‐ene were studied by high‐performance liquid chromatography. It was found that racemization during preparation of N‐(Z‐Pro‐Leu‐)benzoaza‐15‐crown‐5 is promoted by HOBT, an additive usually reducing this process.
Solution conformation of Leu27 hGRF(1‐32)NH2 and its deamidation products by 2D NMR
Authors: STEVENSON CYNTHIA L. , DONLAN MARY E. , FRIEDMAN ALAN R. , BORCHARDT RONALD T.
Published: August 2010

The solution structure and helical content of a human growth hormone releasing factor analog, Leu27 hGRF(1–32)NH2 (hGRF), and its deamidation products Asp8 Leu27 hGRF(l‐32)NH2 and isoAsp8 Leu27 hGRF(1‐32)NH2, were determined by CD and 2D NMR. Chemical‐shift assignments of 1H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn8, Asp8 and isoAsp8 hGRF analogs to have significant α‐helical character. However, the β‐linked isoAsp8 analog did not retain helical structure in the N‐terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn8, Asp8 and isoAsp8 hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N‐terminal region for the isoAsp8 analog when compared with the other two analogs. In the Asn8 and Asp8 hGRF analogs, the helix began at Asp3 or Ala4, while the isoAsp8 analog helix was disrupted until Arg. The higher helicity value for the Asn8 peptide over the isoAsp8 analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn8, Asp8 and isoAsp8 hGRF, respectively.
Chemical modification of ovine prolactin with N‐acetylimidazole
Authors: CYMES GISELA D. , IGLESIAS M. MERCEDES , WOLFENSTEIN‐TODEL CARLOTA
Published: August 2010

Reaction of ovine prolactin (oPRL) with a 150‐fold molar excess of N‐acetylimidazole over protein content resulted in the modification of 2.5 tyrosine residues and 1.2 lysine residues. Acetylation greatly decreased the in vitro binding capacity to lactogenic sites. This binding capacity was partially restored by ammonium bicarbonate treatment, which removes O‐acetyl groups from tyrosine residues but not N‐acetyl groups from lysine residues. The modification extent of the tyrosine residues was determined. The results suggest that acetylation of tyrosine 44 or of tyrosine 96 is likely to be responsible for the decrease in binding activity of acetylated oPRL, and that one of these residues may play a role in the interaction of oPRL with lactogenic receptors.
Interaction ofmyo‐inositol hexaphosphate (MIHP) with β‐globulin fromSesamum indicumL.
Authors: RAJENDRAN S. , PRAKASH V.
Published: August 2010

β‐Globulin, the low molecular weight protein fraction from Sesamum indicum L., interacts with myo‐inositol hexaphosphate (MIHP) maximally at pH 3.0, with concomitant precipitation up to 85 ± 2% at an MIHP concentration of 8 × 10−4m. The kinetics of interaction as followed by stopped‐flow spectrophotometry suggested the reaction to be of pseudo first‐order, having an initial fast step followed by a relatively slow step of rate constants 1.9 × 10−2s−1and 1.2 × 10−2s−1respectively at 1 × 10−4mMIHP concentration. The analysis of the complex indicated the presence of polymer as seen in sedimentation velocity experiment. This was accompanied by conformational change of a three‐fold decrease in β‐structure and also an increase in fluorescence emission intensity accompanied with a red shift from 330 to 334 nm. Stoichiometric analysis of MIHP binding suggested four independent binding sites for MIHP, with a free energy change, ΔGo= ‐5.1 kcal mol−1resulting from a binding constant of 3.6 × 103m‐1.
Design and synthesis of sulfur‐free cyclic hexapeptides which contain the RGD sequence and bind to the fibrinogen GP IIb/IIIa receptor
Authors: LENDER ANDREAS , YAO WENQING , SPRENGELER PAUL A. , SPANEVELLO ROLANDO A. , FURST GEORGE T. , HIRSCHMANN RALPH , SMITH AMOS B.
Published: August 2010

The Arg‐Gly‐Asp (RGD) sequence is the key recognition site in many adhesive interactions. To probe the structural and conformational requirements for potential antithrombotic agents, we have designed and synthesized three cyclic hexapeptides (1, 5and6) containing the RGD sequence. In the ELISA GP IIb/IIIafibrinogen receptor assay,1, 5and6bound with IC50values of 1, 0.1 and 0.016 μm, respectively. All three peptides completely displaced fibrinogen from the receptor. No potent, sulfur‐free cyclic hexapeptide had heretofore been described as a fibrinogen receptor antagonist. The enhanced binding affinity of6, distinguished by the presence of twod‐amino acids, is likely to reflect an increased conformational resemblance to the natural peptide ligands. Cyclization of H‐Asp(OFm)‐dSer‐Phe‐dPhe‐Arg‐Gly‐OH with DPPA and NaHCO3in DMF to afford6was attended by subsequent aspartimide formation with generation of 9‐fluorenylmethanol. Interestingly, imide formation was not observed with any of the three linear hexapeptides (3, 8and9), with the all‐l‐cyclic peptide1, nor with5, which contains only Ser‐1 in the D‐configuration. The observed imide formation led us to use catalytic transfer hydrogenation rather than piperidine to remove the 9‐fluorenylmethyl ester protecting group at the β‐carbonyl of aspartic acid. Further investigation revealed that imide formation was minimized by careful exclusion of water, reducing dissolution of NaHCO3. Thus the distinguishing conformational features of6express themselves both in receptor affinity and chemical propensity toward imide formation.
Synthesis of γ‐carboxyglutamic acid‐containing peptides by the Boc strategy
Authors: NISHIUCHI YUJI , NAKAO MASAYUKI , NAKATA MAKOTO , KIMURA TERUTOSHI , SAKAKIBARA SHUMPEI
Published: August 2010

γ‐Carboxyglutamic acid (Gla) derivatives having several protecting groups at the γ‐carboxyl function were synthesized and examined for their stabilities and removabilities under the conditions used in peptide synthesis by the Boc strategy. Among them, the cyclohexyl (cHx) group of the Gla residue was found to be stable during the synthesis of the protected peptides, but was quantitatively cleaved by the final HF treatment without decarboxylation. Using Boc‐Gla(OcHx)2‐OH as a starting material, the synthesis of Gla‐containing peptides was achieved by the Boc strategy using a standard HF procedure for the final deprotection.
Synthesis and receptor binding analysis of dermorphin hepta‐, hexa‐ and pentapeptide analogues
Authors: ATTILAS M. , SALVADORI S. , BALBONI G. , BRYANT S.D. , LAZARUS L.H.
Published: August 2010

Sixteen dermorphin analogues were synthesized and characterized for μ‐ andδ‐opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or theC‐terminal residue; deletions of Gly4or Pro6‐Ser7; inclusion of Z or an acetyl group on the β‐amino group of Lys7; and the presence of either aC‐terminal amide or free acid group. Two peptides, [Lys7‐OH]‐ and [Lys7‐NH2]dermorphin, had μ‐affinities (Kiμ= 0.15–0.13 nm) and μ‐selectivities (Kiδ/Kiμ= 1158–1482) higher than dermorphin (Kiμ= 0.28 nm;Kiδ/Kiμ= 295) and best fitted a one‐site binding model similar to dermorphin. Significantly better (P<0.0001) fits to a two‐site binding model vs. a one‐site model were observed with four dermorphin analogues: [Lys(Z)7‐OH]heptapeptide, [des‐Gly4(Tyr4,Pro5,Asn6‐OH)]hexapeptide and two pentapeptides, [Tyr5‐NH2] and [Trp4,Asn5‐OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain μ‐receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of μ‐receptors; however, only detailed analyses of the data derived from the non‐linear regression fits for one‐ or two‐components gave evidence for the possible existence of two separate [3H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two‐site model (P<0.05), but only four seemed to have two distinctKi, values (P<0.0001).
Synthesis of a potent antagonist of substance P by replacing the CH2SCH3and the α‐carboxamide groups of the methionine al [Orn6]‐SP6–11by benzyl ester groups
Authors: KARAGIANNIS KOSTAS , STAVROPOULOS GEORGE , POULOS CONSTANTINE , JORDAN CHRISTOPHER C. , HAGAN RUSSELL M.
Published: August 2010

Analogues of [Orn6]‐SP6–11have been synthesized in which the CH2SCH3group of Met11is replaced by a COOCH3or a COOBzl group. These analogues, which were tested for agonist and antagonist activity in threein vitropreparations representative of NK‐1, NK‐2 and NK‐3 receptor types, were full agonists at NK‐1 receptors, showed very weak agonist activity at NK‐2 receptors and were weak antagonists at NK‐3 receptors. The above analogues were modified by substituting the α‐carboxamide of residue 11 by a COOCH3and a COOBzl group, respectively. The resulting analogues were found to be devoid of agonist activity in each of the functional assays. However, they showed weak antagonist activity at each receptor subtype, with the exception of the dibenzyl analogue, which was a potent and selective NK‐1 receptor antagonist. It is concluded that appropriate modification of the side chain of Met11and its α‐carboxamide leads to a potent and selective at NK‐1 receptor antagonist.
Structure and conformation of linear peptides
Authors: SUBRAMANIAN E. , SAHAYAMARY J. JAMES
Published: August 2010

The crystal structure of a dipeptide tert‐butyloxycarbonyl‐l‐alanylglycine monohydrate (C10H18N2O5·H2O), molecular weight 264, has been determined. The crystals are monoclinic, space group P21, with a= 10.767(1), b= 6.317(1), c= 10.981(2) Å, β= 109.15(2)°, and Z= 2, Dc= 1.24 g cm−3. The structure was solved by direct methods and refined to 3 final R‐index of 0.045 for 856 reflections (sin θ/λ < 0.55 Å−1) with I > 2 σ. The N‐terminus of the molecule blocked with the t‐Boc group is uncharged and the C‐terminus exists in an unionized state. The peptide unit is trans and shows slight deviations from planarity. (Δω= 3.1°). The peptide backbone is folded, with torsion angles of φ1= ‐76.0(5), ψ1= 164.3(4), ω1= 176.9(5), φ2= 116.1(5), ψ21= ‐ 2.8(7) and ψ22= 177.8(4)°. The conformation about the urethane bond (C5–N1) is trans. The urethane group is essentially planar. The conformation of the boc group is trans–trans.
Synthesis and immunological properties of bombesin analogs
Authors: CHATURVEDI SANJEEV , PARTHASARATHY R.
Published: August 2010

Bombesin (Bn, pGlu‐Gln‐Arg‐Leu‐Gly‐Asn‐Gln‐Trp‐Ala‐Val‐Gly‐His‐Leu‐Met‐NH2) is one of the most potent peptides, possessing a variety of physiological and pharmacological functions. We find from CD spectroscopy that the eight C‐terminal residues of bombesin [Bn(7‐14)NH2] have an ordered structure, and replacement of His‐12 with Pro of Bn(7‐14)NH2 changes the conformation from ordered to a more unordered form. Antibodies to Bn(7‐14)NH2 cross‐react to Bn and gastrin releasing peptide (GRP) in a dose‐dependent manner. Antibodies to the Pro‐analog do not recognize Bn or GRP. Substitution of the C‐terminal amide by isopropylamide [Bn(7‐14)NHC3H7(i))] makes its antibodies more specific to Bn than to GRP. It appears that this region of the peptide is an important antigenic determinant, which makes these antibodies differentiate between BN and GRP.
Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK‐2 antagonists
Authors: WOLLBORN U. , BRUNNE R.M. , HARTING J. , HÖLZEMANN G. , LEIBFRITZ D.
Published: August 2010

The conformational analysis of three cyclic hexapeptides is presented. Cyclo‐(‐Gln6‐Trp7‐Phe8‐Gly9‐Leu10‐d‐Met11‐) (1) and cyclo‐(‐Gln6‐Trp7‐Phe8‐Gly9‐Leu10‐Met11‐) (2) are NK‐2 antagonists in the hamster trachea assay, whereas cyclo‐(‐Gln6‐Trp7‐Phe8‐(R)‐Gly9‐[ANC‐2]Leu10‐Met11‐) (3), where Gly9[ANC‐2]Leu10 represents (2S)‐2‐((3R)‐3‐amino‐2‐oxo‐1‐pyrrolidinyl)‐4‐methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β‐ and γ‐turns, we find a γn‐turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γi‐turn at Phe8, and βI′‐ or βII‐turns with Gly9 and Leu10 at the corner positions, these β‐turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.
Comparison of 55% TFA/CH2Cl2 and 100% TFA for Boc group removal during solid‐phase peptide synthesis
Authors: BLONDELLE SYLVIE E. , HOUGHTEN RICHARD A
Published: August 2010

Two parallel syntheses of 40 C‐terminal amide peptides, ranging in length from 4 to 20 residues, have been carried out using simultaneous multiple peptide synthesis. All synthetic steps, other than the removal of the Boc group, were performed simultaneously under identical experimental conditions. The two sets of peptides were deprotected with either 55% TFA/DCM for 30 min or 100% TFA for 5 min. The purity of the peptides obtained when deprotecting with 55% TFA/DCM was, on average, 9% higher than with 100% TFA. The major impurity obtained during synthesis when 100% TFA was used for Boc removal corresponded to the omission of the second amino‐acid residue added. Volumetric measurements of the swelling of the resin in the different deprotection solvents were carried out. These showed that the omission analogs generated are probably due to insufficient swelling of the resin, resulting in limited solvent transfer of 100% TFA into the resin and, in turn, incomplete Boc removal.
Surface‐induced conformational switching in amphiphilic peptide segments of apolipoproteins B and E and model peptides†
Authors: TAYLOR JOHN W. , SHIH ING‐LUNG , LEES ANN M. , LEES ROBERT S.
Published: August 2010

The conformational and surface‐binding properties of a synthetic peptide corresponding to Tyr‐apolipoprotein B‐100(1000–1016) amide, SP‐4, which was previously shown to mimic the focal accumulation pattern of LDL on the healing de‐endothelialized rabbit aorta [Shih et al. (1990) Proc. Natl. Acad. Sci. USA87, 1436–1440], have been investigated. SP‐4 behaves as an amphiphilic α‐helical peptide at the air‐water interface and bound to siliconized quartz slides. However, its Nα‐acetylated analogue formed β‐sheet structures at the air‐water interface. Nonhomologous peptide models of SP‐4 also exhibited mixed α‐helical and β‐sheet surface‐binding behavior. Peptides corresponding to the cationic apolipoprotein (apo) B/E receptor binding regions of apoE (SP‐2) and apoB (SP‐11) were also studied. SP‐2 behaved as an amphiphilic α helix, but, surprisingly, SP‐11 formed surface‐induced β‐sheets. These results demonstrate that all of the peptides studied have surface‐binding properties, and suggest further that either α‐helical or β‐sheet peptide structures may determine the binding of LDL to the arterial wall or the apoB/E receptor.
Recombinant human brain‐derived neurotrophic factor (rHuBDNF)
Authors: ACKLIN CHRISTINE , STONEY KENDALL , ROSENFELD ROBERT A. , MILLER JAMES A. , ROHDE MICHAEL F. , HANIU MITSURU
Published: August 2010

Three disulfide linkages of recombinant human brain‐derived neurotrophic factor (BDNF) were determined by peptide sequence analysis and characterized by mass spectrometry. The three disulfide bonds for BDNF expressed in Chinese hamster ovary cells include Cys‐13‐Cys‐80, Cys‐58‐Cys‐109 and Cys‐68‐Cys‐111, and the disulfide structure was homologous to that of nerve growth factor.
Binding of calcium to synthetic peptides containing γ‐carboxyglutamic acid
Authors: COLPITTS TRACEY L. , CASTELLINO FRANCIS J.
Published: August 2010

The Ca2+ binding properties of various γ‐carboxyglutamic acid (gla)‐containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca2+‐selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca2+, with some peptides probably involved in intermolecular bridging of the Ca2+. The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g‐atom of Ca2+ interacts with 2 mol of peptide (n= 0.5) with a Kd value of 1.6 mM. A second g‐atom of Ca2+ interacts with 2 mol of this same peptide (n= 0.5) and is characterized by a Kd of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (n= 2.0)for Ca2+ of Kd=16.1 mM, as well as a tighter site (n= 1), Of Kd= 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38‐residue peptide that possesses all nine of the gla‐residues of protein C in their proper sequence positions. The strongest Ca2+ binding sites (n= 3–4) possess an average Kd of 0.4 mM, followed by another class of sites (n= 5–10, average Kd= 1.5–3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca2+ to the gla region of prothrombin fragment 1. By selective [13C]‐labeling of the essential gla 16 residue of the 38‐mer peptide, we demonstrate that this particular gla residue participates as a donor for a high‐affinity Ca2+ site. These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca2+ to intact gla‐containing proteins.
Multistep deprotection for peptide chemistry
Authors: PATEK MARCEL
Published: August 2010

This review article focusses on some concepts which make use of special classes of protecting groups (protected protecting groups) in peptide synthesis. Terms such as gradative deprotection, dual deprotection, two‐step deprotection, safety‐catch protection and multidetachable protecting systems are discussed, and the general mechanistic considerations for the deprotection process are described.
New method for the synthesis of N‐methyl amino acids containing peptides by reductive methylation of amino groups on the solid phase
Authors: KALJUSTE KALLE , UNDÉN ANDERS
Published: August 2010

Primary amino groups on the model peptide Xaa‐Ala‐Pro‐Lys(ClZ)‐Tyr(2BrZ), synthesized on a p‐methylbenzhydryl amine resin with conventional Boc/benzyl protective group strategy, were reacted with 4,4′‐dimethoxydityl chloride in dichloromethane, resulting in the introduction of the dimethoxydityl group, which is an acid‐labile N‐alkyl type of protective group. The secondary amino groups thereby formed can be methylated by treating the peptide‐resin with formaldehyde and sodium cyanoborohydride in N,N‐dimethylformamide. After the removal of the dimethoxydityl group with trifluoroacetic acid, the resulting N‐methylated amino acid residues with a free secondary amino groups are accessible for acylation with the next activated Boc amino acid. With this method majority of the 20 common amino acids can be monomethylated directly on the resin and, in most cases, with very low levels of the side reactions. In the cases where the complete methylation is difficult to achieve, the remaining primary amino groups can be selectively acylated in the presence of secondary amino groups with trimethylacetic acid 1‐hydroxybenzotriazole ester. The method provides a convenient general route to synthesize N‐methylated derivatives of most of the occurring and synthetic amino acids.
Solid‐phase synthesis of protected peptides using new cobalt(III) ammine linkers†
Authors: ARBO BENTE E. , ISIED STEPHAN S.
Published: August 2010

Cobalt(III) ammine complexes of the type cis‐[CoL4(4‐AMB)O‐AA‐Boc](CF3SO3)2, where L4= bisethylenediamine (en)2 or tetraammine (NH3)4, and 4‐AMB = 4‐(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid‐phase synthesis of protected peptides. Boc/t‐Bu‐protected [Leu5]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC‐purified protected [Leu5]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and 1H NMR. Stepwise synthesis on the Co(en)2‐resin was also used in the assembly of Boc‐Asp(OcHex)‐Arg(Mts)‐Gly‐Asp(OcHex)‐Ala‐Pro‐Lys(2Cl‐Z)‐Gly‐OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC‐purified nonapeptide was characterized by amino acid analysis, 1H NMR and liquid secondary‐ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert‐butyloxycarbonyl (Boc) and the 9‐fluorenylmethoxycarbonyl (Fmoc) Nα‐protecting group strategies used in solid‐phase peptide synthesis.
Superpotent synthetic tripeptide mimics of the mud‐crab pumping pheromone
Authors: PETTIS RONALD J. , ERICKSON BRUCE W. , FORWARD R.B. , RITTSCHOF D.
Published: August 2010

Soluble pheromones released by the mud crab, Rhithropnaopeus harrisii (Gould) during egg hatching cause the female crab to contract her abdomen rapidly (the pumping response). This stereotypical behavior can be induced in the laboratory by exposing egg‐bearing females to solutions containing certain amino acids or peptides. Twelve amino acids exhibited response thresholds of 10−3‐10−10 M, the most potent (norleucine, methionine and tryptophan) having a hydrophobic sidechain. Four synthetic tripeptides of the form Gly‐X‐Arg, where the hydrophobic residue X was isoleucine, norleucine, methionine or phenylalanine, were super‐potent agonists over a limited concentration range. Gly‐Ile‐Arg was a significant agonist at 10−17 and 10−16 M, both Gly‐Nle‐Arg and Gly‐Met‐Arg at 10−20 and 10−19 M, and Gly‐Phe‐Arg at 10−21, 10−20 and 10−19 M. At the subattomolar concentration of 10−20 M the superpotent pheromone mimics Gly‐Met‐Arg and Gly‐Phe‐Arg produced not only a statistically significant increase in the relative number of pumping mud crabs but also a substantial increase in the pumping rate ratio. In contrast, at 10−13 M the tripeptide Gly‐Met(O)‐Arg with an internal residue of methionine sulfoxide blocked the abdominal pumping response of egg‐bearing mud crabs.
Circular dichroism (CD) studies of antagonists derived from parathyroid hormone‐related protein
Authors: CHOREV MICHAEL , EPAND RAQUEL F. , ROSENBLATT MICHAEL , CAULFIELD MICHAEL P. , EPAND RICHARD M.
Published: August 2010

We have undertaken a study of the structure of antagonist peptides derived from the parathyroid hormone‐related protein (PTHrP) in the presence of amphiphiles using circular dichroism (CD). The results were used to gain knowledge about bioactive conformations of the peptide when bound to a membrane. The substitutions within the PTHrP‐(7‐34)amide sequence resulted in differences in biological activity. Structural determination by CD showed the presence of an α‐helical structure. The antagonist activity was increased in constrained peptides in which i to (i+ 4) side‐chain to side‐chain cyclization was used to form a lactam,[Lys13,Asp17]PTHrP‐(7‐34)NH2. This peptide showed increased helicity in the presence of a surfactant. Hydrophobic substitutions Leu and d‐Trp at positions 11 (Lys) and 12 (Gly), respectively, in PTHrP‐(7‐34)NH2 resulted in increased potency, but the derivatives were not significantly more helical than the un‐substituted peptide in the presence of surfactants. The combination of the hydrophobic substitutions with the constraint of lactam formation were mutually exclusive in terms of their biological activity and their α‐helical content. We conclude that hydrophobic substitutions contribute to an increase in binding affinity by increasing hydrophobic interactions which stabilize receptor‐ligand complexes. Structural rigidification, on the other hand, increases the α‐helical content, which is important for attaining a conformation recognized by the receptor.
Systematic study of substance P analogs
Authors: WANG JIAN‐XIN , BRAY ANDREW M. , DIPASQUALE ANGELA J. , MAEJI N. JOE , GEYSEN H. MARIO
Published: August 2010

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large‐scale screening of oligopeptides [Geysenet al.(1984)Proc. Natl. Acad. Sci. USA, 81, 3998‐40021. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated.

Substance P and 18 analogs, including a set ofN‐terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 ± 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against125I‐BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high‐volume screening of bioactive peptides.
Efficient Fmoc/solid‐phase synthesis of Abu(P)‐containing peptides using Fmoc‐Abu(PO3Me2)‐OH
Authors: PERICH JOHN W.
Published: August 2010

The synthesis of the two 4‐phosphono‐2‐aminobutanoyl‐containing peptides, Leu‐Arg‐Arg‐Val‐Abu(P)‐Leu‐Gly‐OH.CF3CO2H and Ile‐Val‐Pro‐Asn‐Abu(P)‐Val‐Glu‐Glu‐OH.CF3CO2H was accomplished by the use of Fmoc‐Abu(PO3Me2)‐OH in Fmoc solid‐phase peptide synthesis. The protected phosphoamino acid, Fmoc‐Abu(PO3Me2)‐OH, was prepared from Boc‐Asp‐O'Bu in seven steps, the formation of the C—P linkage being effected by the treatment of Boc‐Asa‐O'Bu with dimethyl trimethylsilyl phosphite. Peptide synthesis was performed using Wang Resin as the polymer support with both peptides assembled by the use of PyBOP® for the coupling of Fmoc amino acids and 20%, piperidine for cleavage of the Fmoc group from the Fmoc‐peptide after each coupling cycle. Cleavage of the peptide from the resin and peptide deprotection was accomplished by the treatment of the peptide‐resin with 5%, thioanisole/TFA followed by cleavage of the methyl phosphonate group by 1 M bromotrimethylsilane/l M thioanisole in TFA.
Comparing conformations at low temperature and at high viscosity
Authors: VERHEYDEN PATRICIA , DE WOLF ELKE , JASPERS HENDRIKA , VAN BINST GEORGES
Published: August 2010

The influence of low temperature and high viscosity on the conformation of somatostatin and two of its analogues was investigated by 1H NMR in solution. The conformation of native somatostatin, a cyclic octapeptide agonist DC13‐116 and a linear octapeptide agonist were compared in ethylene glycol at 303 K and in methanol at low temperature. The first goal of this study was to investigate if either low temperature or high viscosity is the more important for the reduction of the conformational freedom. Secondly we wanted to compare the amount of information concerning the conformation present in both solvents. A larger amount of NOESY cross‐peaks is observed in ethylene glycol at room temperature compared to methanol at low temperature. This indicates that the raising of the viscosity is a more important factor in reducing the flexibility of peptides than the lowering of the temperature.
N‐Cyclohexyl‐N′ ‐isopropylcarbodiimide: a hybrid that combines the structural features of DCC and DIC
Authors: IZDEBSKI JAN , PACHULSKA MARIA , ORLOWSKA ALICJA
Published: August 2010

N‐Cyclohexyl‐N′ ‐isopropylcarbodiimide was prepared and used for peptide couplings by various procedures. Reaction between Z‐Val and Gly‐OEt was used for yield studies and determination of the extent of the side reaction in solution. The value of this reagent for solid peptide synthesis was demonstrated by the synthesis of sequences comprising the 65–74 residues of acyl carrier protein. The efficiency of activation was determined by competition experiments. From all these studies the conclusion can be drawn that N‐cyclohexyl‐N′‐isopropylcarbodiimide is comparable to or even better than the commonly used DCC for mediation of peptide bond formation in solid phase synthesis. By virtue of the solubility of N‐cyclohexyl‐N′‐isopropylurea in dichloromethane, the carbodiimide seems to be a good candidate for practical execution of peptide bond formation by the standard carbodiimide procedure.
Substitution of aromatic and nonaromatic amino acids for the Phe3residue in the δ‐selective opioid peptide deltorphin I: Effects on binding affinity and selectivity
Authors: HEYL DEBORAH L. , SCHMITTER SHARON J. , BOUZIT HASSIBA , JOHNSON THOMAS W. , HEPP ANGELA M. , KURTZ KATHLEEN R. , MOUSIGIAN CAROL
Published: August 2010

Deltorphins I and II (Tyr‐D‐Ala‐Phe‐Asp‐Val‐Val‐Gly NH2and Tyr‐D‐Ala‐Phe‐Glu‐Val‐Val‐Gly NH2) display a high degree of 6‐opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapep‐tides based on Tyr‐c[D‐Cys‐Phe‐D‐Pen] (JOM‐13). With the goal of development of structure‐activity relationships for deltorphins and comparison with that of the cyclic tetrapep‐tides, ten analogs of deltorphin I were synthesized in which Phe3was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3‐(l‐naphthyl)‐L‐alanine [1‐Nal;Ki(μ) = 767 nM,Ki(§) = 7.70 nM], 3‐(2‐naphthyl)‐L‐alanine [2‐Nal; Ki(μ)= 1910 nM, Ki(§) = 49.2 nM], tryptophan [Ki(μ)= 1250 nM, Ki(§) = 23.9nM], and 3‐(3‐benzothienyl)‐L‐alanine [Bth;Ki(μ)= 112nM,Ki(§) = 3.36 nM] were fairly well tolerated at μ‐ and §‐receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both μ (Ki= 4710 nM) and § (Ki= 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced μ binding (Ki= 67.8 nM), leaving § affinity unaffected (Ki= 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [Ki(μ) =≥ 10000 nM for each,Ki(§) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both μ (Ki= 322 and 1240 nM, respectively) and § (Ki= 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr‐c[D‐Cys‐Phe‐D‐Pen].
p‐Chlorotetrafluorophenyl esters of N‐protected amino acids
Authors: MEDVEDKIN VIACHESLAV N. , KLIMENKO LIUBOV N. , MITIN YURI V. , KRETSINGER ROBERT H. , SHABANOWITZ JEFFREY , ZABOLOTSKIKH VASILII F. , PODGORNOVA NATALIA N.
Published: August 2010

p‐Chlorotetrafluorophenyl (Tfc) esters of protected amino acids and peptides are more reactive than are the well known pentafluorophenyl (Pfp) esters. Two reagents, p‐chlorotetrafluorophenyltrifluoroacetate (Tfc‐OTfa) and di‐(p‐chlorotetrafluorophenyl)carbonate (di‐Tfc‐carbonate), can be used for their syntheses, thereby avoiding use of the allergic dicyclohexylcarbodiimide. This is especially important for bulk preparations. Many Fmoc‐ and Boc‐amino acid‐OTfc esters have been synthesized and characterized. The hexadecameric tanden1 repeat H‐(AlaAlaLysPro)4‐OH was synthesized using di‐Tfc‐carbonate for the preparation of Tfc‐esters.
Hydration and distortion of peptide helices in crystals. α‐Helical structure of a dodecapeptide, Boc‐(Ala‐Leu‐Aib)4‐OMe
Authors: KARLE ISABELLA L. , FLIPPEN‐ANDERSON JUDITH L. , UMA K. , BALARAM P.
Published: August 2010

The dodecapeptide Boc‐(Ala‐Leu‐Aib)4‐OMe crystallized with two independent helical molecules in a triclinic cell. The two molecules are very similar in conformation, with a 310‐helix turn at the N‐terminus followed by an α‐helix, except for an elongated N(7)…O(3) distance in both molecules. All the helices in the crystal pack in a parallel motif. Eleven water sites have been found in the head‐to‐tail region between the apolar helices that participate in peptide‐water hydrogen bonds and a network of water‐water hydrogen bonds. The crystal parameters are as follows: 2(C58H104N12O15) +ca. 10H2O, space group P1 with a= 12.946(2), b= 17.321(3), c= 20.465(4)Å, α=103.12(2), β= 105.63(2), γ= 107.50(2)˚, Z= 2, R= 10.9% for 5152 data observed > 3σ(F), resolution 1.0 Å. In contrast to the shorter sequences [Karle et al. (1988) Proc. Natl. Acad. Sci. USA85, 299–3031 and Boc‐(Ala‐Leu‐Aib)2‐OMe [Karle et al. (1989) Biopolymers28, 773–781], no insertion of a water molecule into the helix is observed. However, the elongated N—O distance between Ala7 NH and Aib3 CO in both molecules (molecule A, 3.40 Å; molecule B, 3.42 Å) is indicative of an incipient break in the helices.
Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium
Authors: PORTA R. , ESPOSITO C. , SCHININA M.E. , MANCUSO F. , MARINO G. , PUCCI P. , PELUSO G. , METAFORA S.
Published: August 2010

Two fragments of SV‐IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met‐70) occurring in its polypeptide chain. After their purification by reversed‐phase chromatography, SV‐IV/A (71‐70 fragment) and SV‐IV/B (71–90 fragment) were assayed as transglutaminase substrates, and their anti‐inflammatory, anti‐thrombotic and immunosuppressive properties were evaluated in comparison with native SV‐IV. Both fragments retained the SV‐IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln‐9 and Gln‐86 as acyl donor sites, and to Lys‐59, ‐79 and ‐80 as acyl acceptor sites. In contrast, only SV‐IV/A was shown to possess, like SV‐IV, the property of inhibiting both the intensity of the carrageenin‐induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen‐induced proliferation of human T cells and the mixed lymphocyte reaction.
Efficient Fmoc/solid‐phase peptide synthesis of O‐phosphotyrosyl‐containing peptides and their use as phosphatase substrates
Authors: PERICH JOHN W. , RUZZENE MARIA , PINNA LORENZO A. , REYNOLDS ERIC C.
Published: August 2010

A general synthetic method for the efficient preparation of Tyr(P) ‐containing peptides is described by the use of Fmoc‐Tyr(PO31Bu2) ‐OH in Fmoc/solid‐phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H‐Ser‐Ser‐Ser‐Tyr(P) ‐Tyr(P) ‐OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8–13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T‐cell protein tyrosine phosphatase. In a competition assay using 32P‐radiolabeled [Tyr(P)]4‐angiotensin II, both un‐labeled synthetic [Tyr(P)]4‐angiotensin II and Ser‐Ser‐Ser‐Tyr(P) ‐Tyr(P) reduced the release of 32P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]4‐neurotensin 8–13 was ineffective as a competitive substrate and indicated that this particular Tyr(P) ‐containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp‐Arg‐Val‐Tyr(P) ‐Ile‐His‐Pro‐Phe and Arg‐Arg‐Pro‐Tyr(P) ‐Ile‐Leu is consistent with the presence of an acidic residue in the ‐3 position relative to the Tyr(P) residue.