Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin.

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA and H-Glu-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA were prepared by the use of Boc-Ser(PO3Ph2)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO3Ph2)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH2Cl2 used for removal of the Boc group from intermediate Boc-protected peptides.     

Further studies on the use of 2,2,2-trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis.

Boc-Ser(PO3Tc2)-OH, Z-Ser(PO3Tc2)-OH and Fmoc-Ser(PO3Tc2)-OH, derivatives useful for peptide synthesis, have been obtained in high yields by acylation of H-Ser(PO3Tc2)-OH.CF3COOH. The latter was obtained from Boc- or Z-Ser(PO3Tc2)-OBzl by simultaneous removal of the amino- and carboxy-protecting groups by Pd-catalyzed hydrogenolysis in acetic acid-trifluoroacetic acid solution. Removal of the Tc-protecting group was efficiently achieved by hydrogenolysis in aqueous ethanol.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-glucopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an alpha or beta O-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[alpha-Glc(OBzl)4]-OBzl and Z-Thr[alpha-Gal(OBzl)4]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The alpha glycosylated threonine derivatives were converted into Fmoc-Thr(alpha-Glc)-OH and Fmoc-Thr(alpha-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. beta-Glucosylation and beta-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the beta-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(beta-Glc)-OH and Fmoc-Thr(beta-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO2)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and 1H NMR. The beta-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo[Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)2, and cyclo(Glu-Pro)2, were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)-Pro]2 and cyclo(Glu-Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.     

Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.     

Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester as a novel potential PET gene reporter probe for HBV and HSV-tk in cancers

Acyclic nucleoside 2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (ABE, 1) is a new hepatitis B virus (HBV) specific antiviral reagent and shows high anti-HBV activity. Carbon-11 labeled ABE may serve as a novel reporter probe for positron emission tomography (PET) to image HBV and herpes simplex virus thymidine kinase (HSV-tk) in cancers. The radiolabeling precursors 2-amino-6-(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (10) and 2-N-Boc protected analogue 2-N-bis(Boc)amino-6-(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (12), and the reference standard ABE were synthesized from bis(trifluoroethyl) (2-iodoethoxy)methylphosphonate (5), guanine (6), and 2-amino-6-chloropurine (8). The target radiotracer 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE, [11C]1) was prepared by O-[11C]methylation of the unprotected HO-precursor 10, or 2-N-Boc protected HO-precursor 12 with [11C]methyl triflate followed by a quick deprotection reaction, and isolated by solid-phase extraction (SPE) purification in 40-55% radiochemical yields.     

Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

A solid-phase synthetic strategy for the preparation of peptide-based affinity labels: synthesis of dynorphin A analogs.

Solid-phase synthetic methodology was developed for the preparation of peptide-based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p-X)4,D-Pro10]Dyn A(1-11)NH2 containing isothiocyanate (X=-N=C=S) and bromoacetamide (X=-NHCOCH2Br) groups. The peptides were assembled on solid supports using Fmoc-protected amino acids, and the side chain amine to be functionalized, Phe(p-NH2), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(O), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol-polystyrene (PEG-PS) resins containing the PAL [peptide amide linker, 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity-labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

Specificity of cathepsin B to fluorescent substrates containing benzyl side-chain-substituted amino acids at P1 subsite

We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures NH₂-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (ortho-aminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBzl), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH₂Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S₁ subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in substrates containing Cys(SBzl) amino acid at P₁ seems to compensate the absence of adequate S₂-P₂ interaction in the hydrolysis of the peptides having Pro or Ala at P₂ position.     

'O-Acyl isopeptide method' for peptide synthesis: Solvent effects in the synthesis of Abeta1-42 isopeptide using 'O-acyl isodipeptide unit'.

'O-Acyl isopeptide method' is an efficient synthetic method for peptides. We designed 'O-acyl isodipeptide units', Boc-Ser/Thr(Fmoc-Xaa)-OH, as important building blocks to enable routine use of the O-acyl isopeptide method. In the synthesis of an Abeta1-42 isopeptide using O-acyl isodipeptide unit Boc-Ser(Fmoc-Gly)-OH, a side reaction, resulting in the deletion of Ser(26) in the O-acyl isopeptide structure, was noticed during coupling of the unit. We observed that the side reaction occurred during the activation step and was solvent-dependent. In DMF or NMP, an intramolecular side reaction, originating from the activated species of the unit, occurred during the activation step. In non-polar solvents such as CHCl(3) or CH(2)Cl(2), the side reaction was less likely to occur. Using CH(2)Cl(2) as solvent in coupling the unit, the target Abeta1-42 isopeptide was synthesized with almost no major side reaction.     

Relationship between conformation and physicochemical properties of polypeptides. I. Synthesis of homo- and co-oligopeptides by the liquid-phase method

The synthesis of the following oligo- and co-oligopeptides by the liquid-phase method is described: (L -Met)₁₅ (I), [L -Glu(OBzl)]₂₀ (II), (L -Val)₈-Gly (IV), (L -Ile)₈-Gly (V), (L -Ile)₄-Gly-(L -Ile)₄ (VI), (L -Ile)₄-Pro-(L -Ile)₄ (VII), (L -Met)₅-L -Pro-(L -Met)₅ (VIII), [L -Glu(OBzl)]₇-L -Pro-[L -Glu(OBzl)]₇ (IX). The oligomers are covalently bound to bifunctional polyethylene glycol (PEG) and monofunctional PEG-M of M_r 5 × 10³?2 × 10⁴. Analytical controls were carried out after each step of synthesis in order to ensure quantitative coupling yields. All products could be obtained in high purity as indicated by amino acid analysis, thin-layer chromatography and chiroptical methods. The solubility of the oligomers was strongly enhanced by the presence of the C-terminal PEG group, enabling conformational investigations in a variety of solvents. A significant relationship between conformation and physicochemical properties of the oligopeptides was observed. Oligomers with tendencies to adopt α-helical (I, II) or unordered structures (VI–IX) showed no pronounced change in solubility or coupling kinetics during chain elongation, whereas the onset of a β-structure (IV, V) was paralleled by a drastic decrease in solubility and reactivity of the terminal amino groups. Most notably, the insertion of a proline or glycine in the middle of a β-forming peptide chain (VI, VII) resulted in a considerable increase in solubility compared to the corresponding homo-oligomers. The impact of the conformational properties of a peptide chain on strategic considerations of peptide synthesis in solution is delineated.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

The synthesis of alternative diketopiperazines as potential RGD mimetics.

Alternative RGD mimetics-with the exception of glycine-c(Arg-Asp) 1, c(Arg-Glu) 2 and c[Arg-Asp(Phe-OH)] 3 were synthesized. The DKPs were prepared on solid phase with orthogonal protection allowing further derivatization in solution. During solution phase cyclization in NH(3)/methanol, the side chain benzyl ester group of H-Arg(Tos)-Asp(OBzl)-OMe and H-Arg(Tos)-Glu(OBzl)-OMe suffer transesterification, while beta-t-butyl or beta-cyclohexyl esters are stable under the same conditions. In spite of the simple structure, all compounds bind selectively to the alpha(v)beta(3) integrin receptor, 3 showing the highest affinity with an IC(50) value of 0.74 microM value. On the other hand only 3 binds with measurable activity to the alpha(IIb)beta(3) receptor (IC(50) 159 microM). The binding affinities seem to be in accordance with the distances between the arginine guanidino and the aspartic acid carboxyl group in extended conformation determined by semiempirical geometry optimization.     

Peptide models of electrostatic interactions in proteins: NMR studies on two beta-turn tetrapeptides containing Asp-His and Asp-Lys salt bridges

Two model peptides Boc-Asp-Pro-Aib-X-NHMe [X = His (1) and X = Lys (2)] were synthesized to simulate intramolecular electrostatic interactions between ionizable side chains. Conformational analysis by 270-MHz 1H NMR in (CD3)2SO reveals that the backbone secondary structures of these two peptides are stabilized by two strong intramolecular hydrogen bonds, involving the consecutive carboxy-terminal NH groups. 1H NMR chemical shifts were measured in 1, 2, and a protected derivative, Boc-Asp(OBzl)-Pro-Aib-His-NHMe (3). These shifts were also measured for the model compounds Ac-Lys-NHMe, Boc-Asp-NHMe, and Boc-His-NHMe in their different states of ionization. An analysis of the chemical shifts of the ionization-sensitive reporter resonances suggests the formation of a strong intramolecular salt bridge in the lysyl peptide 2 and a bridge of moderate strength in the histidyl peptide 1. A comparison of the temperature dependence of chemical shifts in peptides 1-3 suggests that intramolecular salt bridge formation results in diminished backbone flexibility. The results establish that proximity effects confer far greater stability to intramolecular ion pair interactions vis-a-vis their intermolecular counterparts. The salt bridge interaction in peptide 1 displays a remarkable sensitivity to the dielectric constant of the solvent medium. The results suggest that these peptides are good simulators of the role of salt bridges in the structural dynamics of proteins.     

Tuftsin and some analogs: synthesis and interaction with human polymorphonuclear leukocytes

The phagocytosis-stimulating tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, was synthesized by both conventional and polymeric-reagent approaches. Using a combination of the two methods several analogs were prepared, including: [Ala1]tuftsin, [Lys1]tuftsin, [Ser1]tuftsin, [Val1]tuftsin, acetyl-tuftsin, p-aminophenylacetyl-tuftsin and tyrosyl-tuftsin. [Des-Thr1]tuftsin and [omega-NO2(4)]tuftsin were synthesized using a conventional procedure. The effects of synthetic peptides on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes were investigated. Tuftsin and to a lesser extent [Lys1]tuftsin and [Ser1]tuftsin were found to stimulate phagocytosis, whereas the other analogs synthesized as well as [Ser1]tuftsin exhibited inhibitory effects to tuftsin's action. Tuftsin alone has stimulated nitroblue tetrazolium reduction; [Des-Thr1]tuftsin and [Ala1]tuftsin repressed this stimulation, while the other peptides showed no effect.     

Synthesis of linear,cyclic and polypeptides having the sequence -Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly- as catalysts in hydrolytic reactions

Boc-Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly-OEt, Boc-Asp-βAla-Gly-Ser-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly-OH, cyclo(Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly) and poly(Asp-βAla-Gly-Ser-βAla-Gly-His-βAla-Gly) were synthesized as catalysts in hydrolytic reactions. Asp and Ser residues were protected with a benzyl group, but the His residue was not protected during the synthesis. The protected linear-nonapeptides were prepared by synthesis and subsequent fragment condensation of three kinds of tripeptide which have the sequences-Asp(OBzl)-βAla-Gly-,-Ser(Bzl)-βAla-Gly- and -His-βAla-Gly-, respectively. The protected cyclic-nonapeptide was obtained by cyclization of H-Asp(OBzl)-βAla-Gly-Ser(Bzl)-βAla-Gly-His-βAla-Gly-OH with DCC-HOBr and the subsequent purification by silica gel column chromatography. The protected poly-nonapeptide was prepared by polymerization of H-Asp(OBzl)-βAla-Gly-Ser(Bzl)-βAla-Gly-His-βAla-Gly-OH with DPPA. Deprotection was performed with catalytic hydrogenation for the protected linear- and cyclic-nonapeptides, and with methanesulphonic acid for the protected polynonapeptide, respectively, to give final products. Catalytic actions of the linear-, cyclic- and polypeptides for hydrolysis of p-nitrophenyl acetate are also reported briefly.     

Synthesis by conventional methods of human growth hormone peptide fragments. I.

The synthesis of two protected peptides which correspond to positions 139-146 and 147-156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Asn-Ser(Bzl)-His(Dnp)-Asn(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

Molecular and crystal structures of Aib-containing oligopeptides Boc-Leu4-Aib-Leu4-OBzl and Boc-(Leu4-Aib)2-OBzl.

Sample peptides Boc-Leu4-Aib-Leu4-OBzl and Boc-(Leu4-Aib)2-OBzl, were crystallized by the solvent-evaporation method. Both crystals are monoclinic, with space group of P2(1) and Z = 2. The cell parameters are a = 16.580 (7), b = 21.105 (7), c = 11.583 (4) A, and beta = 104.90 (3) degrees (Boc-Leu4-Aib-Leu4-OBzl), and a = 15.247 (9), b = 19.04 (1), c = 16.311 (9) A, and beta = 117.10 (1) degrees [Boc-(Leu4-Aib)2-OBzl]. Crystal structures were solved by the direct method and refined to R values of 0.096 (the former peptide) and 0.112 (the latter). Peptide backbones fold into a right-handed alpha-helix, except for the C-terminal Aib residue in Boc-(Leu4-Aib)2-OBzl. Both peptide molecules are stabilized by six (the former) or seven (the latter) intramolecular (5----1) hydrogen bonds, and arranged in the head-to-tail fashion, which makes an infinite column. In this column, one (the former) or two (the latter) intermolecular hydrogen bonds link the neighboring molecules. In the case of Boc-Leu4-Aib-Leu4-OBzl, the solvent molecule N,N-dimethylformamide was found in the difference Fourier map. There was a hydrogen bond between peptide and solvent molecule. Along the lateral direction, only hydrophobic contacts were observed between adjacent peptide molecules.