Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Poly(L-lysyl-L-alanyl-alpha-L-glutamic acid). II. Conformational studies.

The solution characterization of poly(Lys-Ala-Glu) is described. This polytripeptide is zwitterionic at neutral pH and is shown to take on a conformation which is dictated by the state of ionization, molecular weight, temperature, and solvent. The polypeptide is almost entirely α-helical at low pH and temperature for polymers of greater than 25,000 molecular weight. Melting profiles for these conditions show t_m ～ 20°C. Analysis of circular dichroism curves shows the α-helical content to vary in a linear manner with molecular weight in the range 3000–30,000. At neutral pH the charged polypeptide is essentially random, but substantial α-helix could be induced by addition of methanol or trifluoroethanol. At temperatures where the sequential polypeptide is a random coil, addition of trifluoroethanol produces a polymer which is mostly α-helical but also contains an appreciable ammount of β-structure. The infrared spectrum of a low-molecular-weight fraction assumed to be cyclo(Lys-Ala-Glu)₂ was tentatively assigned a β-pleated sheet structure. A comparison of this polytripeptide in various ionization states with other polytripeptides containing L -alanine and L -glutamate or L -lysine shows the α-helix directing properties for the (uncharged) residues to lie in the order Ala > Glu > Lys.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Optical properties of cyclic dimers of amino acids: An experimental and theoretical study

CD spectra have been measured in the vacuum-uv region to about 135 nm for cyclo(L alanyl-glycine), cyclo(L alanyl-L -alanine), and cyclo(L -prolyl-L -proline). In addition to the amide CD bands usually observed at wavelengths longer than 180 nm, a Independent systems calculations show that these intense short-wavelenght bands can be attributed to σσ* transitions of the backbone. Transitions of the amide chromophore expected at wavelenghts shorter than 180 nm cannot account for the observed CD.     

The optical properties of tyrosine-containing cyclic dipeptides

The chiroptical properties of the cyclic dipeptides cyclo (L -alanyl-L -tyrosine) and cyclo(L -tyrosyl-L -tyrosine) have been investigated as a function of molecular conformation. Theoretical optical calculations and conformational energy calculations have been carried out as a function of the side-chain dihedral angles χ¹ and χ², and as a function of the angle of fold of the cyclic dipeptide backbone. The results of these theoretical calculations have been compared with experimental circular dichroism (CD) data. Theoretical predictions are in very good agreement with experiment for c(L -Tyr-L -Tyr). Agreement was not quite as good for c(L -Ala-L -Tyr), although the signs of all of the Cotton effects were apparently predicted correctly except for that associated with the lowest energy tyrosine absorption band.     

Synthesis and conformational studies of peptides containing TOAC,a spin-labelled Cα,α-disubstituted glycine

A variety of host L -alanine homo-peptides (to the pentamer) containing one or two spin-labelled TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) residues were synthesized by solution methods and fully characterized. The conformational features of the terminally blocked, doubly spin-labelled–TOAC–(Ala)₂–TOAC–Ala– pentapeptide were examined in the crystal state by X-ray diffraction and in solution using a combination of techniques (Fourier transform infrared, circular dichroism, cyclic voltammetry and electron spin resonance) in comparison with singly labelled shorter peptides. The 3₁₀-helical structure of the pentapeptide, promoted by the two C^α,α-disubstituted glycines under favourable experimental conditions, allows an interaction to take place between the two nitroxide TOAC side chains spaced by one turn of the helix. Taken together, these results suggest that TOAC is an excellent probe for exploring bends and helices in doubly labelled peptides.     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Polymerization of L- and DL-phenylalanine N-carboxyanhydride by poly(N-methyl-L-alanine)

Polymerizations of L - and DL -phenylalanine N-carboxyanhydride in nitrobenzene by poly (N-methyl-L -alanine) of varying degrees of polymerization (n = 1–30) were investigated. Poly(N-methyl-L -alanine) was prepared by the polymerization of N-methyl-L -alanine NCA with N-methyl-L -alanine diethylamide and the degree of polymerization was controlled by the molar ratio [NCA]/[Catalyst] + 1. This polymer was shown to be an asymmetrically selective catalyst which polymerized L -phenylalanine NCA at a faster rate than DL -phenylalanine NCA. With increasing degree of polymerization the stability of the secondary structure of poly(N-methyl-L -alanine) increased. This was confirmed by circular dichroism spectra. However, the degree of asymmetric selection did not increase as the stability of the secondary structure of poly(N-methyl-L -alanine) increased. These findings indicate that the interaction of a growing polypeptide in an ordered structure with NCA molecules prior to the reaction does not lead to an asymmetric selection, and that the mechanism of the asymmetric selection by poly(N-methyl-L -alanine) should be different from those proposed so far.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Vibrational CD studies of the solution conformation of N-urethanyl-L-amino acid derivatives

The solution conformations of several N-urethanyl-L -amino acids in 0.2–0.002M chloroform and 0.2M dimethylsulfoxide have been investigated by using vibrational circular dichroism (VCD), ir absorption, and ¹³C-nmr spectroscopies. Both the N-carbobenzoxy (N-CBZ) and N-t-butoxycarbonyl derivatives of L -alanine, L -proline, L -valine, and L -phenylalanine, and N-CBZ-serine were studied. The ¹³C-nmr results indicate that the molecules occur predominantly as the cis isomer about the C? N peptide bond at room temperature. The interpretation of the strong VCD couplet in the carbonyl-stretching region using the degenerate coupled oscillator model is consistent with the conformational range of ? = ?60° to ?90° and ψ = 30° to 90° for molecules forming acid dimers. Conformations with an intramolecularly hydrogen-bonded C₇ ring are also present, which give rise to biased VCD features in the NH-, CH-, and C?O-stretching regions that can arise due to vibrationally generated ring currents.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular modeling and design of regioselectively addressable functionalized templates with rigidified three‐dimensional structures

Extensive conformational analysis of a series of β‐alkyl substituted cyclopeptides—cyclo(Pro¹–Xaa²–Nle³–Ala⁴–Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Ala⁹–Nle¹⁰) and cyclo[Pro¹–Xaa²–Nle³–(Cys⁴– Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Cys⁹)–Nle¹⁰] as well as their corresponding unsubstituted core structures cyclo(Pro¹–Xaa²–Ala³–Ala⁴–Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Ala⁹–Ala¹⁰) and cyclo(Pro¹–Xaa²–Ala³–Cys⁴– Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Cys⁹–Ala¹⁰) has been performed employing both the ECEPP/2 and the MAB force fields (Xaa = Gly, L ‐Ala, D ‐Ala, Aib, and D ‐Pro). Results show that (a) possible three‐dimensional structures of the cyclo(Pro¹–Gly²–Lys³–Ala⁴–Lys⁵–Pro⁶–Gly⁷–Lys⁸–Ala⁹–Lys¹⁰) molecule are not limited to a single extended “rectangular” conformation with all Lys side chains oriented at the same side of the molecule; (b) conformational equilibrium in monocyclic analogues obtained by replacements of conformationally flexible Gly residues for L ‐Ala, D ‐Ala, Aib, or D ‐Pro is not significantly shifted towards the target “rectangular” conformational type; and (c) introduction of disulfide bridges between positions 4 and 9 is a very powerful way to stabilize the target conformations in the resulting bicyclic molecules. These findings form the basis for further design of rigidified regioselectively addressable functionalized templates with many application areas ranging from biostructural to diagnostic purposes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 361–372, 1999     

Influence of D and L amino-acid residues on the conformation of peptides in solution: A carbon-13 nuclear magnetic resonance study of cyclo(prolyl-leucyl)

The ¹³C chemical shifts and spin-lattice relaxation times are reported for cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu). The chemical shifts of the D and L leucyl residues in the cyclic peptides differ from each other by 1.8 and 3.6 parts per million for the α and β carbons, respectively. The α-carbons of the prolyl residues differ by 1.0 ppm as a consequence of proximity to a D or an L leucyl residue. The ¹³C spin-lattic relaxation time(T₁) of the prolyl residues, but not the leucyl residues, in both compounds are indicative of difference in conformational equilibria within the pyrrolidine ring in the L -L isomer as compared to the L -D isomer. Anisotropic overall molecular reorientation is not responsible for the differences observed in the T₁ values. The differences in T₁ values and chemical shifts between cyclo(L -Pro-L -Leu) and cyclo(L -Pro-D -Leu) appear to result from a difference in conformations of the two diketopiperazine rings.     

Characterization of (Boc‐Cys/Sec‐NHMe)2 and (Boc‐Cys/Sec‐OMe)2: Evidence of local conformational difference between disulfide and diselenide

Conformations of disulfide and diselenide were compared in (Boc‐Cys/Sec‐NHMe)₂ and (Boc‐Cys/Sec‐OMe)₂ using X‐ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT), and circular dichroism (CD) spectroscopy. Conformations of disulfide/diselenide in polypeptides are defined based on the sign of side chain torsion angle χ₃ (–CH₂–S/Se–S/Se–CH₂–); negative indicates left‐handed and positive indicates right‐handed orientation. In the crystals of (Boc‐Cys‐OMe)₂ and (Boc‐Sec‐OMe)₂, the disulfide exhibits a left‐handed and the diselenide a right‐handed orientation. Characterization of cystine and selenocystine derivatives in solution using ¹H‐NMR, natural abundant ⁷⁷Se NMR, 2D‐ROESY, and chemical shift analysis coupled to DMSO titration has indicated the symmetrical nature and antiparallel orientation of Cys/Sec residues about the disulfide/diselenide bridges. Structural calculations of cystine and selenocystine derivatives using DFT further support the antiparallel orientation of Cys/Sec residues about disulfide/diselenide. The far‐ultraviolet (UV) region CD spectra of cystine and selenocystine derivatives have exhibited the negative Cotton effect (CE) for disulfide and positive for diselenide confirming the difference in the conformational preference of disulfide and diselenide. In the previously reported polymorphic structure of (Boc‐Sec‐OMe)₂, the diselenide has right‐handed orientation. In the X‐ray structures of disulfide and diselenide analogues of Escherichia coli protein encoded by curli specific gene C (CgsC) retrieved from Protein Databank (PDB), disulfide has left‐handed and the diselenide right‐handed orientation. The current report provides the evidence for the local conformational difference between a disulfide and a diselenide group under unconstrained conditions, which may be useful for the rational replacement of disulfide by diselenide in polypeptide chains.     

Comprehensive Chiroptical Study of Proline‐Containing Diamide Compounds

The continuously growing interest in the understanding of peptide folding led to the conformational investigation of methylamides of N‐acetyl‐amino acids as diamide models. Here we report the results of detailed conformational analysis on Ac‐Pro‐NHMe and Ac‐β‐HPro‐NHMe diamides. These compounds were analyzed by experimental and computational methods, the conformational distributions obtained by Density Functional Theory (DFT) calculations for isolated and solvated diamide compounds are discussed. The conformational preference of proline‐containing diamide compounds as a function of the ambience was observed by a number of chiroptical spectroscopic techniques, such as vibrational circular dichroism (VCD), electronic circular dichroism (ECD), Raman optical activity (ROA) spectroscopy, and additionally by single crystal X‐ray diffraction analyses. Based on a comparison between Ac‐Pro‐NHMe and Ac‐β‐HPro‐NHMe, one can conclude that due to the greater conformational freedom of the β‐HPro derivative, Ac‐β‐HPro‐NHMe shows different behavior in solid‐ and solution‐phase, as well. Ac‐β‐HPro‐NHMe tends to form cis Ac‐β‐HPro amide conformation in water, dichloromethane, and acetonitrile in contrast to its α‐Pro analog. On the other hand, the crystal structure of the β‐HPro compound cannot be related to any of the conformers obtained in vacuum and solution while the X‐ray structure of Ac‐Pro‐NHMe was identified as tα_L–, which is a trans Ac‐Pro amide containing conformer also predominant in polar solvents. Chirality 26:228–242, 2014. © 2014 Wiley Periodicals, Inc.     

Evidence from circular dichroism for the binding of hydrogen ions and calcium ions by poly (L-proline).

The positive circular dichroism band observed near 228 nm with poly(L -proline) responds in a similar fashion to HCl and CaCl₂. The spectra in the HCl solutions are compatible with a simple binding equation and a pK near ?2 for the dissociation of a proton from a protonated peptide bond in poly(L -proline). The data obtained in CaCl₂ is susceptible to the same analysis, suggesting a pK near ?1.5 for the dissociation of a calcium ion from its complex with poly(L -proline).     

Ascidiacyclamides containing oxazoline and thiazole motifs assume square conformations and show high cytotoxicity

Four cyclic octapeptides were designed from ascidiacyclamide [cyclo(–Ile–Oxz–D ‐Val– Thz–)₂] (ASC, 1 ) to investigate the effects of oxazoline (Oxz) and thiazole (Thz) rings on the structures and cytotoxicities of the peptides. cyclo(–Ile–Thz–D ‐Val–Oxz–)₂ ( 2 ) had the same number of Oxz and Thz rings as ASC, but the ring positions were switched. cyclo(–Ile–Oxz–D ‐Val–Thz–Ile–Thz–D ‐Val–Thz–) ( 3 ) and cyclo(–Ile–Thz–D ‐Val–Oxz–Ile–Thz–D ‐Val–Thz–) ( 4 ) contained one Oxz and three Thz rings within the molecule. All Oxz rings were substituted with Thz in cyclo(–Ile–Thz–D ‐Val–Thz–)₂ ( 5 ). These analogues had new Oxz and Thz blocks forming the 24‐membered ring. Based on CD spectra and X‐ray diffraction analyses, the structures of all four analogues were classified as square ASC forms. But the structures of 2 and 5 differed from the original square form of 1 , and they showed no cytotoxicity. The structure of 3 was very similar to that of 1 , and 3 showed 10 times greater cytotoxicity than 1 . Although no definite structure of 4 was obtained, it showed three times greater cytotoxicity than 1 . It appears that the position and number of Oxz residues are essential determinants in the structure‐cytotoxicity relationship of ASC analogues.