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

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

Racemization during aminolysis of mixed and symmetrical anhydrides of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide

Racemization attending the aminolysis of symmetrical (Sy) and mixed (Mx) anhydrides (An) of N-alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide has been examined by reversed phase high-performance liquid chromatography analysis of the N-protected dipeptides produced. Racemization occurred in most cases when the anion was generated using 1 equiv. sodium hydrogen carbonate, in most cases for MxAn reactions when the base was sodium carbonate, and only in a few cases for SyAn reactions when the base was sodium carbonate. Less racemization was associated with MxAn reactions when the chloroformate of a secondary alcohol had been used for their generation. The change in chirality is explained on the basis of the formation and racemization of the 2-alkoxy-5(4H)-oxazolone, which is greater in the presence of the weaker base because of incomplete deprotonation of the amino acid zwitter-ion.     

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

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

Practical protocols for stepwise solid‐phase synthesis of cysteine‐containing peptides

Abstract: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62 , 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH₂, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH₂Cl₂/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluenesulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Identification and suppression of decomposition during carbodiimide-mediated reactions of Boc-amino acids with phenols,hydroxylamines and amino acid ester hydrochlorides

N-tert-Butoxycarbonylamino acids (Boc-Xaa-OH) were coupled with p-nitrophenol (HONp) in dichloromethane using N,N′-dicyclohexylcarbodiimide (DCC) and N-ethyl-N′(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the products were identified and quantitated by high-performance liquid chromatography. Boc-Xaa-OH with Xaa = Val was coupled also with pentafluorophenol (HOPf) and hydroxy-containing additives (HOR), and the products were similarly determined as the methylamides. EDC-mediated reactions of Boc-Xaa-OH gave 8–25% of Boc-Xaa-Xaa-OR as well as Boc-Xaa-OR for R = Ph, Np, Pf, benzotriazole (Bt) and 5-norbornene-endo-2,3-dicarboxamide; DCC-mediated couplings, 5–7% for R = Np and Bt. No dimer was formed in couplings with N-hydroxysuccinimide or 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine. Dimerization was eliminated from DCC-mediated reactions by the addition of 1 equiv. of N-methylmorpholine, from the EDC-mediated reactions by carrying them out in pyridine. Dimerization is attributed to formation of the intermediate 2-alkoxy-5(4H)-oxazolone that undergoes fragmentation to the N-carboxyanhydride, which reacts with the alcohol giving amino acid ester. Ester produces dimer by aminolysis of the O-acylisourea. Decomposition (1.4%) was also detected by analysis for H-Val-Phe-OMe in DCC-mediated reactions of Boc-valine with H-Phe-OMe, and was demonstrated to be caused by the hydrochloride of the ester salt that had been neutralized with N-methylmorpholine. Decomposition was eliminated by the addition of 5%, of pyridine, which also had the beneficial effect of suppressing N-acylurea formation.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine‐containing fMLF analogs

Abstract: In order to explore the properties of chemotactic N‐formylpeptides containing isopeptide bonds within their backbones, a group of lysine‐containing analogs of the prototypical chemotactic tripeptide N‐formylmethionyl‐leucyl‐phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO‐Met or Boc‐Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N^α or N^ε bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO‐Met‐Lys(Z)‐Phe‐OMe ( 2a , b ), RCO‐Met‐Lys(Z‐Phe)‐OMe ( 3a , b ), Z‐Lys(RCO‐Met)‐Phe‐OMe ( 4a , b ), Z‐Phe‐Lys(RCO‐Met)‐OMe ( 5a , b ), RCO‐Met‐Phe‐Lys(Z)‐OMe ( 6a , b ) and Z‐Lys(RCO‐Met‐Phe)‐OMe ( 7a , b ), with R=OC(CH₃)₃ and R=H for compounds a and b , respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b ) and antagonists (compounds a ). All N‐formyl derivatives 2b ? 7b are less potent than fMLF‐OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a ? 7a do not show antagonistic activity towards fMLF induced chemotaxis and O₂^? production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Pseudopeptides containing the 2‐hydrazonoacyl fragment: analogs of the chemotactic agent HCO‐Met‐Leu‐Phe‐OMe

Abstract: In order to further examine the properties of pseudopeptides containing the 2‐hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N‐formyl‐tripeptide HCO‐Met‐Leu‐Phe‐OMe were designed and synthesized. The first group contains the new fragment as the N‐terminal residue and is represented by the N‐aryl derivatives p‐Cl‐C₆H₄‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 2 and 3 ) and by the corresponding N‐aroyl analogs p‐CH₃‐C₆H₄‐CO‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 4 ). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO‐Xaa‐NH‐N=C(R)‐CO‐Phe‐OMe ( 7a and 7b ) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.     

Conformational study on trans- and cis-N-acetyl-N′-methylamides of Pro-Xaa dipeptides

Conformational free energy calculations using an empirical potential ( ECEPP /2) and the hydration shell model were carried out on the N-acetyl-N′-methylamides of Pro-Xaa dipeptides (Xaa = Ala, Leu, Val, Gly, Cys, Met, Phe, Tyr, Asn, Asp, and Ser) with trans and cis peptide bonds preceding proline residue in the unhydrated and hydrated states. As compared with the results obtained by using the earlier version of ECEPP, the values of β-bend probabilities are about doubled. The average calculated population of cis-dipeptide is about 4%, which is close to the abundance obtained from the analysis of X-ray crystal structures of proteins. The β-bends are the most dominant structures of cis-dipeptides. Type I, usually having intramolecular hydrogen bonds, contributes greatly to the β-bend conformations of trans- and cis-dipeptides. However, type I β-bends of cis-dipeptides do not have any hydrogen bonds. By including the hydration, the β-bend probabilities for trans- and cis-dipeptides decreased, indicating that the interactions of water molecules with a backbone or side-chain may force the dipeptides to be more distorted or extended. In particular, type II is found to be a dominant β-bend conformation of trans- and cis-Pro-Gly dipeptides in both the unhydrated and hydrated states. In general, the calculated propensities for Pro-Xaa dipeptides to adopt β-bend conformations are reasonably consistent with available experimental data. From comparing conformations of Pro and Xaa residues in the dipeptides and single residues, we found that inter-residue interactions and hydration are of importance in determining the conformational properties of the Pro-Xaa dipeptide.     

Cyclic morphiceptin analogs: cyclization studies and opioid activities in vitro

Attempts were undertaken to develop cyclic β-casomorphin-5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH₂). The tetrapeptide sequence Boc-Tyr(tBu)-D-Xaa-Phe-Yaa-OH (Xaa = Lys, Orn, A₂bu; Yaa = Pro in L- or D-configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the ω-amino group of Xaa and the C-terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and ¹H-NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L-configuration could a cyclic monomer be detected, and the cyclodimer ( 7–9 ) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11-membered ring of the cyclic [D-A₂bu², D-Pro⁴]-morphiceptin analog 12 , since only traces of the monomer were found. In the case of both the [D-Lys², D-Pro⁴]-analog 10 and the [D-Orn², D-Pro⁴]- analog 11 , the cyclomonomer/cyclodimer ratio was about 80: 20. The cyclic monomers 10 and 11 showed high opioid activity in the μ-receptor-representative guinea pig ileum assay (IC₅₀= 2–5 nM) and in the δ-receptor representative mouse vas deferens assay (IC₅₀= 50–60 nM), whereas the potency of the cyclodimers was 2–3 orders of magnitude lower in both in vitro bioassays.     

The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides

Peptide-4-nitroanilides can be quickly synthesised using an Fmoc-based approach on 2-chlorotritylchloride resin. Preformed building blocks Fmoc-Xaa-NH-Np (Xaa = Cit, Cys, Gin, His, Lys, Orn, Ser, Thr, Tyr, Trp) can be attached via side chain to the 2-chlorotritylchloride linker of the resin. N-terminal elongation yields the respective peptide-4-nitroanilides after detachment from the solid support. We synthesised a set of tetrapeptide-4-nitroanilides with the general structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Asp, Cit, Cys, Glu, Gin, His, Lys, Orn, Ser, Thr, Tyr, Trp). Even peptidyl-arginine-4-nitroanilides are available by a slightly modified procedure. First, the appropriate ornithine-containing peptide was synthesised. After detachment of the peptide from the resin the side-chain primary amino group was transformed to the guanidino function of arginine using 1-guanyl-3, 5-dimethylpyrazole. A further application of this method is the convenient synthesis of phosphorylated peptide-4-nitroanilides. Five phosphopeptides with the general structure Ac-Ala-Xaa(PO₃H₂)-Pro-Yaa-NH-Np (Xaa = Ser, Thr, Tyr; Yaa = Tyr, Lys) and their nonphosphorylated analogues were prepared. Global phosphorylation was carried out on the resin-bound peptides using dibenzyl-N, N-diisopropyl-phosphoramidite/tetrazole followed by oxidation with tert-butyl hydroperoxide. © Munksgaard 1997.     

Kinetic study of racemization of aspartyl residues in model peptides of αA-crystallin

We have reported that two aspartyl (Asp-151 and Asp-58) residues in αA-crystallin in human eye lens were inverted to the D-isomer and isomerized to β-aspartyl residues with age. We report here the kinetics of the Asp racemization of three model peptides corresponding to fragments of αA-crystallin: IQTGLD¹⁵¹ATHAER (T18 peptide). TVLD⁵⁸SGISEVR (T6 peptide) and HFSPED⁸⁴LTVK (T10 peptide, as a control). The rate constants of the racemization of Asp residues in these peptides were measured at pH 7.0. at five temperatures: 50, 60, 70, 80 and 90 °C. From the Arrhenius equation, we estimated the activation energy (E) of racemization and the time required for the Asp D/L ratio to approximate to 1.0 (D/L ratio of Asp = 0.99) at body temperature. For the peptide T18, E=21.4 kcal/mol and t=13.5 yr. For the peptide T6, E= 26.8 kcal/mol and t= 49.5 yr. For the control peptide T10, E=28.3 kcal/mol and t= 78.1 yr. The racemization rate of Asp in these three peptides is parallel to that of Asp residues in αA-crystallin. The racemization rate of Asp in the T18 peptide was very rapid compared to that in the other peptides. This result also reflects the racemization rate in native αA-crystallin     

Lipoconjugates: structure—activity studies for pheromone analogues of Ustilago maydis with varied lipophilicity

The synthesis, biological activities and conformational behaviour of a variety of analogues of the mating pheromones of the basidomycete Ustilago maydis are reported. The pheromone analogues derived from the two allelic forms H-G-R-D-N-G-S-P-I-G-Y-S-S-Xaa-Z (a1) and H-N-R-G-Q-P-G-Y-Y-Xaa-Z (a2), with Xaa-Z being an unidentified lipophilic cysteine derivative, all differ in the C-terminal residue and include -Cys(farnesyl)-OMe, -Cys(farnesyl)-OH, -Cys(prenyl)-OMe, -Cys-OMe, -Cys(n-dodecyl)-OMe and the unnatural residues -Ahds-OMe (Ahds =α-aminohexadecanoic acid), -Ahds-OH, -Ads-OMe (Ads =α-aminodecanoic acid) and -N-Hdg-OMe (N-Hdg=N-hexadecylglycine). The synthesis of the unnatural methyl ester analogues was carried out by condensation of the fully protected fragments Fmoc-G-R(Pmc)-D(tBu)-N(Trt)-G-S(tBu)-P-I-G-Y(tBu)-S(tBu)-S(tBu)-OH (a1′) and Fmoc-N(Trt)-R(Pmc)-G-Q(Trt)-P-G-Y(tBu)-Y(tBu)-OH (a2′) respectively, prepared by Fmoc-SPPS, with the appropriate methylester compounds and subsequent deprotection with TFA/scavenger and piperidine. Synthesis and physicochemical properties of the unnatural lipophilic amino acid methylesters are described. The preparation of the cysteine analogues was performed by condensation of a1′ or a2′ with H-Cys(Trt)-OMe and subsequent deprotection with TFA/scavenger. Alkylation of the thiol function and Fmoc-deprotection was achieved in a novel one-pot reaction by treatment with alkyl bromide and DIPEA, quenching with EDT and Fmoc removal by addition of 20% piperidine (v/v). Hydrolysis of the methyl esters was carried out by treatment with NaOH in MeOH/H₂O. The results of the biological assay reveal an increase in activity with increasing chain length of the lipophilic anchor, with alkyl being better than prenyl and sulfur being not essential, while the position of the anchor is optimal at Cα and the methyl ester moiety is important. NMR studies of two chosen analogues in DMSO and SDS/water demonstrate that the lipophilic C-terminal residue has no influence on the structural behaviour of the peptides. Chemical-shift and NOE patterns indicate a main all-trans conformation of the peptide backbone and a weakly populated cis conformation around the Xaa-Pro peptide bond in all eight cases without formation of a defined folded structure. No evidence is seen that the membrane-simulating system SDS/water has a structure-inducing effect on the bound peptide. We therefore conclude that the lipomodification in mating pheromones of U. maydis acts to increase the effective concentration of the drug in the target cell membrane without additional structure-inducing or receptor-binding effects. © Munksgaard 1996.     

Side-product formation during cyclization with HBTU on a solid support

The coupling reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was used in an attempt to prepare a highly strained 10-membered lactam ring on a solid support via side-chain to side-chain cyclization of the adjacent α, γ-diaminobutyric (Dab) and d -glutamic acid residues in [Dab²,d -Glu³,Leu⁵]enkephalinamide. This attempted cyclization failed, however, and yielded linear products instead. Characterization by mass spectrometry, amino acid analysis, peptide sequencing and NMR indicated that the major products were the tetramethylguanidinium (Tmg) derivatives [Dab(Tmg)²,d -Glu³,Leu⁵]-enkephalinamide and the corresponding dimeric linear Tmg-containing peptide which resulted from the transfer of the tetramethyluronium moiety from HBTU to the amino side chain of Dab. The formation of these tetramethylguanidinium side products during cyclization reactions limits HBTU's usefulness for the formation of lactams.     

Crystal structure of L-lysyl-L-glutamic acid dihydrate

L-Lysyl-L-glutamic acid dihydrate, C₁₁N₃O₅H₂₁·2H₂O, crystallizes in the monoclinic space group P2₁ with a = 12.474(2), b = 5.020(1), c = 13.157(2) Å, β= 114.69(1)° and Z = 2. The crystal structure was solved by direct methods and refined to an R value of 0.037 using full matrix least-squares method. The molecule exists as a double zwitterion with both the amino and carboxyl groups ionised. The peptide has a folded conformation with its Lys residue trans and Glu residue gauche^?gauche⁺. The side chains of the Lys and Glu residues correspond to all trans and folded (g^?g^?g^?) conformations respectively. The terminal carboxyl group forms hydrogen bonds with the ξ-amino group of the lysine side chain. The head-to-tail interaction often seen in peptide crystals is absent in the present structure. In the extended crystal structure water molecules form channels along the b direction and are enclosed within helically arranged hydrogen bonds formed by the lysine side chain and the peptide backbone.     

Solid-phase synthesis of H- and methylphosphonopeptides

We introduce solid-phase syntheses of H- and methylphosphonopeptides, giving access for the first time to a new class of mimics for o-phosphoamino acids. The model peptides H-GlyGlyXaaAla-OH (Xaa = Ser, Thr) were synthesized on a solid-phase using Fmoc/^tBu strategy and HBTU/HOBt activation by incorporation of hydroxyl-protected serine and threonine. As selectively cleavable hydroxyl-protecting groups we used triphenylmethyl and tert-butyldimethylsilyl for both amino acids, as described in the literature. All peptides were phosphitilated with O,O-di-tert-butyl-N,N-diethylphosphoramidite and yielded H-phosphonopeptides after trifluoroacetic acid cleavage. Alternatively we phosphonylated the peptides with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite, which was synthesized by a two-step one-pot procedure starting from commercially available chemicals. All H- and methylphosphonopeptides were obtained in high purities and yields, as shown by reversed-phase high-performance liquid chromatography and anion-exchange chromatography. The phosphonopeptides were characterized by ¹H and ³¹P NMR. We confirmed their molecular masses by electrospray mass spectrometry and analyzed their fragmentation schemes, which seemed to be characteristic for each class of analogues. The H-phosphonopeptides lost phosphonic acid (H₃PO₃, 82 mass units) and the methylphosphonopeptides lost methylphosphonic acid (MeH₂PO₃, 96 mass units). Both H- and methylphosphonopeptides represent a new and simply accessible class of mimics for phosphopeptides. Compared with the corresponding phosphopeptides all phosphonopeptides were synthesized in higher yields and purities (>80%). © Munksgaard 1996.     

Studies on asymmetric induction associated with the coupling of N-acylamino acids and N-benzyloxycarbonyldipeptides

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)-oxazolones obtained from N-acyl-dl -valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and A′-benzyloxycarbonyl-Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at + 5° and – 5° was determined by analysis of the epimeric products by high-performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side-chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)-oxazolones from N-benzyloxycar-bonyl giycyl-Xaa-OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N′-dicyclohexylcarbodiimide in the presence and absence of 1-hydroxybenzotriazole, and by the mixed-anhydride method, were calculated. The induction was affected at varying levels by the nature of the N-acyl group, the side-chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching – 32 and +53. The influence of the side-chain of Xaa on the induction was different in the two solvents. For the N-acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.     

The crystal structure of Boc-Pro-Val-Gly-NH2, with a remark on the conformation of the valyl residues in peptides

The crystal structure of Boc-Pro-Val-Gly-NH₂ has been determined: monoclinic; P2₁; a = 9.331 (3) Å, b = 9.532 (4), c = 23.080 (9), β= 91.33 (3)R, Z = 4; R = 0.053 for 3400 reflections with ˙Fo˙,>α(Fo). There are two independent but very similar molecules in the crystal. The peptide main chains are in an extended form, and packed in two kinds of antiparallel β sheets, the (φ, Φ) angles of the central Val residues are (-156°, 146°) and (-139°, 155°), and the mean length of the N- H . 0 hydrogen bonds in the sheets is 2.965 Å. A detailed study of the conformations of the Val residues in oligopeptide crystals shows that the preferred conformation of Val in peptides is: the (φ, Φ) angles close to those of the antiparallel β sheet, and C^γ1 and C^γ2, against N with respect to the C^α– C^β bond, at either (trans, gauche) or (-gauche, gauche). The mean π(NC^αC') angle of such Val residues is 107.9(9)°. A twisting in the β sheets is also discussed.     

Aza-peptides. III. Experimental structural analysis of aza-alanme and aza-asparagine-containing peptides

To determine the structural perturbations induced by the CαH→Nα exchange in aza-peptides, we have examined by H NMR and IR spectroscopy various derivatives of the aza-analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R'-AzXaa-NR²R³, R'-Pro-AzXaa-NR²R³ and R-AzXaa-Pro-NR²R³ (where AzXaa denotes the aza-analogue of the amino acid residue Xaa = Ala, Asp, Asn; R = Boc, Z; R², R³= H, Me, iPr). The aza-analogue of an amino acid residue appears to be a strong p-turn-inducing motif, and the AzAsn carboxamide side-chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.     

The Problem of Racemization in the Stereospecific Assay and Pharmacokinetic Evaluation of Ketorolac in Human and Rats

Purpose. A comparison of a previously reported indirect (precolumn derivatization) assay for ketorolac (KT) and a new direct method described here was made to establish the conditions under which KT may undergo racemization and to explain the observed discrepancies in the pharmacokinetics of KT reported in the literature. Methods. A previously reported pre-column derivatization method and a new direct method were employed to determine the effect of pH and ionic strength on racemization. Using the conditions where no racemization occurred, the pharmacokinetics in humans and rats, and protein binding of KT enantiomers were determined. Results. Under the chromatographic conditions employed for the direct assay, no racemization was observed. Under high pH and ionic strength, however, both methods resulted in KT racemization. The indirect method resulted in rapid and complete racemization due to the strong basic conditions required for derivatization. In both humans and rats, the pharmacokinetics of racemic KT were stereoselective with the R enantiomer being predominant (AUC S/R: humans, 0.26; Rats: 0.45). This is likely due to more extensive plasma protein binding of S than its antipode (unbound S/R: 1.35). Conclusions. The discrepancies in the literature may be explained by rapid racemization of KT that occurs during sample preparation for the pre-column derivatization method. Considerations should be given to the possibility of racemization during the assay development and validation.