Linoleic acid peroxidation vs. isomerization: a biomimetic model of free radical reactivity in the presence of thiols

Biomimetic models of free radical-induced transformation of polyunsaturated fatty acids, such as micelles and liposomes, have been used for the study of lipid peroxidation and lipid isomerization. Free radical reactivity of thiol compounds is the common link between the two processes, since lipid peroxidation is inhibited by thiols, due to their H-donation ability, whereas lipid isomerization is catalysed by S-centered radicals. In this paper the two processes are compared for the first time, in solution and under biomimetic conditions, demonstrating that hydroperoxides and trans lipids are formed to comparable extents as a result of oxidative free radical conditions. The biomimetic model of micelles of linoleic acid, prepared by addition of a non-ionic surfactant (TWEEN(?)-20) and 2-mercaptoethanol as the amphiphilic thiol, was irradiated by ionizing radiation up to 400 Gy under various conditions. In air-equilibrated solutions, the cis-trans isomerization process was observed with a catalytic cycle of 370 together with a substantial amount of hydroperoxides (LOOH). The effect of micelle size was also studied in order to envisage the effect of the supramolecular organization on the outcome of the two processes, and in particular, for the positional preference of the double bond isomerization.     

Cis-trans isomerization of chemically activated 1-methylallyl radical and fate of the resulting 2-buten-1-peroxy radical

The cis-trans isomerization of chemically activated 1-methylallyl is investigated using RRKM/Master Equation methods for a range of pressures and temperatures. This system is a prototype for a large range of allylic radicals formed from highly exothermic (～35 kcal/mol) OH + alkene reactions. Energies, vibrational frequencies, anharmonic constants, and the torsional potential of the methyl group are computed with density functional theory for both isomers and the transition state connecting them. Chemically activated radicals are found to undergo rapid cis-trans isomerization leading to stabilization of significant amounts of both isomers. In addition, the thermal rate constant for trans → cis isomerization of 1-methylallyl is computed to be high enough to dominate reaction with O(2) in 10 atm of air at 700 K, so models of the chemistry of the (more abundant and more commonly studied) trans-alkenes may need to be modified to include the cis isomers of the corresponding allylic radicals. Addition of molecular oxygen to 1-methylallyl radical can form 2-butene-1-peroxy radical (CH(3)CH═CHCH(2)OO(?)), and quantum chemistry is used to thoroughly explore the possible unimolecular reactions of the cis and trans isomers of this radical. The cis isomer of the 2-butene-1-peroxy radical has the lowest barrier (via 1,6 H-shift) to further reaction, but this barrier appears to be too high to compete with loss of O(2).     

Regioselective cis-trans isomerization of arachidonic double bonds by thiyl radicals: the influence of phospholipid supramolecular organization

Trans unsaturated fatty acids in humans may be originated by two different contributions. The exogenous track is due to dietary supplementation of trans fats and the endogenous path deals with free-radical-catalyzed cis-trans isomerization of fatty acids. Arachidonic acid residue (5c,8c,11c,14c-20:4), which has only two out of the four double bonds deriving from the diet, was used to differentiate the two paths and to assess the importance of a radical reaction. A detailed study on the formation of trans phospholipids catalyzed by the HOCH2CH2S* radical was carried out on L-alpha-phosphatidylcholine from egg lecithin and 1-stearoyl-2-arachidonoyl-L-alpha-phosphatidylcholine (SAPC) in homogeneous solution or in large unilamellar vesicles (LUVET). Thiyl radicals were generated from the corresponding thiol by either gamma-irradiation or UV photolysis, and the reaction course was followed by GC, Ag/TLC, and 13C NMR analyses. The isomerization was found to be independent of cis double bond location (random process) in i-PrOH solution. In the case of vesicles, the supramolecular organization of lipids produced a dramatic change of the isomerization outcome: (i) in egg lecithin, the reactivity of arachidonate moieties is higher than that of oleate and linoleate residues, (ii) in the linoleate residues of egg lecithin, the 9t,12c-18:2 isomer prevailed on the 9c,12t-18:2 isomer (3:1 ratio), and (iii) a regioselective isomerization of SAPC arachidonate residues occurred in the 5 and 8 positions. This effect of "positional preference" indicates that thiyl radicals entering the hydrophobic region of the membrane bilayer start to isomerize polyunsaturated fatty acid residues having the double bonds nearest to the membrane surfaces. We propose that arachidonic acid and its trans isomers can function as biomarkers in membranes for distinguishing the two trans fatty acid-forming pathways.     

Direct calculation of interconversion barriers in dynamic chromatography and electrophoresis: Isomerization of captopril

Dynamic capillary electrophoresis (DCE) and direct calculation of the rate constants of isomerization has been applied to determine the cis-trans isomerization barriers of the angiotensin-converting enzyme inhibitor captopril. The separation of the rotational cis-trans isomeric drug has been performed in an aqueous 50 mM borate buffer at pH 9.3. Interconversion profiles featuring plateau formation, peak-broadening, and peak coalescence were observed. To determine the rate constants of the forward and backward reaction (k(cis-->trans) and k(trans-->cis)) of the isomerization process in dynamic capillary electrophoresis, a novel straightforward calculation method using the experimental parameters plateau height, h(plateau), peak width at half height w(h), the total migration times of the cis-trans isomers t(R) and the electroosmotic break-through time t(0) as well as the peak ratio of the cis-trans isomers is presented for the first time. From temperature dependent measurements the rate constants k(cis-->trans) and k(trans-->cis) and the kinetic activation parameters DeltaG( not equal), DeltaH( not equal), and DeltaS( not equal) of the cis-trans isomerization of captopril were obtained. From the activation parameters the isomerization barriers of captopril at 37 degrees C under basic conditions were calculated to be DeltaG( not equal) (cis-->trans) = 90.3 kJ.mol(-1)and DeltaG( not equal) (trans-->cis) = 90.0 kJ.mol(-1*).     

Low temperature high-performance liquid chromatography of cis-trans proline dipeptides

Isomeric dipeptides which contain proline at the C-terminus and undergo on-column cis-trans isomerization were separated by reversed-phase chromatography at subambient column temperature. Chromatography at low temperatures offers a convenient method for measuring the relative concentrations of the isomers and, thus, the calculation of the equilibrium constants for such cis-trans isomerization at various conditions of solvent, pH, and temperature. Pure fractions of the cis and trans isomers were collected and used in kinetic studies of the isomerization. High-performance liquid chromatography at temperatures near the freezing point of the eluent is a potentially useful tool for the study of a wide range of biochemical molecules.     

Determination of the cis-trans isomerization barrier of several L-peptidyl-L-proline dipeptides by dynamic capillary electrophoresis and computer simulation

Dynamic capillary electrophoresis (DCE) and computer simulation of the elution profiles with the theoretical plate and the stochastic model has been applied to determine the isomerization barriers of the three dipeptides L-alanyl-L-proline, L-leucyl-L-proline, and L-phenylalanyl-L-proline. The separation of the rotational cis-trans isomers has been performed in an aqueous 70 mM borate buffer at pH 9.5. Interconversion profiles featuring plateau formation and peak broadening were observed. To determine the rate constants k1 and k(-1) of the cis-trans isomerization in dynamic capillary electrophoresis, equations have been derived for the theoretical plate model and stochastic model. The electropherograms were simulated with the ChromWin software which uses the experimental data plateau height h(plateau), peak width at half height Wh, the total migration times of the cis-trans isomers tR and the electroosmotic break-through time t0 as well as the peak ratio [cis]/[trans]. From temperature-dependent measurements, the rate constants k1 and k(-1) and the kinetic activation parameters deltaG#, deltaH# and deltaS# of the cis-trans isomerization of the three dipeptides were obtained.     

Determination of the cis-trans isomerization barrier of enalaprilat by dynamic capillary electrophoresis and computer simulation

Dynamic capillary electrophoresis (DCE) and computer simulation of the elution profiles with the stochastic model has been applied to determine the isomerization barriers of the angiotensin converting enzyme inhibitor enalaprilat. The separation of the rotational cis-trans isomeric drug has been performed in an aqueous 20 mM borate buffer at pH 9.3. Interconversion profiles featuring plateau formation and peak broadening were observed. To evaluate the rate constants k(cis-->trans) and k(trans-->cis) of the cis-trans isomerization from the experimental electropherograms obtained by dynamic capillary electrophoresis, elution profiles were analyzed by a simulation with iterative convergence to the experimental data using the ChromWin software which requires the total migration times of the individual isomers t(R), the electroosmotic break-through time t(0), the plateau height h(plateau), the peak widths at half height of the individual isomers w(h), as well as the peak ratio of the isomers as experimental data input. From temperature-dependent measurements between 0 degrees and 15 degrees C the thermodynamic parameters Delta G, Delta H and Delta S, the rate constants k(cis-->trans) and k(trans-->cis) and the kinetic activation parameters Delta G*, Delta H*, and Delta S* of the cis-trans isomerization of enalaprilat were obtained. From the activation parameters the isomerization barriers at 37 degrees C were calculated to be Delta G* (trans-->cis) = 87.2 kJ.mol(-1) and Delta G*(cis-->trans) = 91.9 kJ.mol(-1).     

脂肪酸的色谱保留时间规律与质谱特征研究及其在食品分析中的应用

用石油醚提取食品中的脂肪,经甲酯化反应后,采用HP-88(100m×0.25mm,0.33μm)弹性石英毛细管柱分离脂肪酸甲酯的同系物及异构体,GC/MS法测定。研究了不同链长脂肪酸的同系物及异构体的气相色谱出峰顺序,得到其保留时间规律;研究了不同脂肪酸的质谱断裂规律,选择3个特征离子来鉴定脂肪酸成分。建立了3个特征离子确定脂肪酸碳数及双键数目,色谱保留时间规律确定脂肪酸顺反异构体及双键位置异构体的方法。本法无需标准品即可快速测定脂肪酸同系物及异构体的含量,适用于脂肪酸组成的研究;及油脂、食品中脂肪酸,特别是反式脂肪酸的测定。     

Analysis of conjugated linoleic acids as 9-anthrylmethyl esters by reversed-phase high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method for determining the fatty acid composition of food lipids containing conjugated linoleic acid (CLA) is described. The method is based on the separation of the 9-anthrylmethyl ester derivatives of saturated and unsaturated (conjugated and non-conjugated) fatty acids by reversed-phase high-performance liquid chromatography with fluorescence detection. Just like the other fatty acids, CLA reacts readily with 9-anthryldiazomethane at room temperature to produce 9-anthrylmethyl esters without isomerization and decomposition of the conjugated double bonds. Clear resolution of the individual fatty acids as their 9-anthrylmethyl esters is achieved on a highly efficient octadecylsilylated silica column (150- x 3-mm i.d., 3-microm particle size) using a stepwise gradient elution with methanol-water. The method is standardized with commercially available CLA isomers (cis-9, trans-11 and trans-10, cis-12-octadecadienoic acids, and their cis,cis and trans,trans isomers) and applied for determination of the fatty acid compositions of milk and sdairy products.     

Separation of cis/trans geometrical fatty acid isomers by silver-exchanged zeolite Y

The separation of cis and trans isomers is relevant for biological and nutritional applications, silver-exchanged zeolite Y was prepared and applied for the treatment of mixtures of cis and trans geometrical isomers of mono- and polyunsaturated fatty acid methyl esters (FAMEs). cis FAMEs were adsorbed into zeolite with a high degree of selectivity (cis/trans ratio in the range of 1.9-3.2). The effectiveness was due to the synergism of the π-complexation between the silver ion and the double bonds and the different FAME structures trapped into the zeolite cages. Some indication of the complex stabilities came from theoretical studies using unsaturated lipids. A prototype cartridge was also designed for application in the fractionation of cis/trans FAME mixtures.     

Photoisomerization of arylstilbazolium ligands in the presence of DNA

The ability of the DNA duplex to behave as an efficient organized medium for trans-cis photoisomerization has been explored. The presence of DNA affected isomerization in a variety of ways depending on the aryl moiety properties of the ligand and its DNA-binding mode. Contrary to intercalating ligands, 9-[2-(N-methylpyridinium-4-yl)vinyl]phenanthrene (2) and 9-[2-(N-methylpyridinium-4-yl)vinyl]anthracene (3), which gave only cis and trans isomers, the additional products--cyclobutane photodimers--were detected for 2-[2-(N-methylpyridinium-4-yl)vinyl]naphthalene (1), which binds cooperatively to the minor groove of DNA. Photostationary states (pss) for all ligands were seriously affected by the presence of DNA. A trans isomer-rich pss and restriction of trans --> cis process, observed for ligands 1 and 2, were explained in terms of a different binding affinity of DNA toward particular isomers. On the contrary, 9-anthryl derivative 3 isomerized against the isomer-binding preferences, showing cis isomer-rich pss and enhanced quantum yield of isomerization. The unique behavior of ligand 3-DNA complex was attributed to different isomerization mechanism that consists in quantum chain isomerization from an excited singlet state possessing a charge transfer character. This is the first example of ligand, which undergoes DNA-mediated cis-trans isomerization in the opposite direction than expected from DNA-binding preferences. The analysis of pss data based on two alternative pathways of photoisomerization showed that investigated ligands follow the model that allows isomerization of both free and DNA-bound ligands.     

Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation

Reaction efficiencies of two organic alkalis, tetramethylammonium hydroxide (TMAH) and trimethylsulfonium hydroxide (TMSH), with lipids during thermally assisted hydrolysis and methylation (THM) were examined focusing on (1) the types of lipids and (2) degree of unsaturation of fatty acid moieties. Different types of lipids such as triglycerides, phospholipids, free fatty acids and cholesteryl esters containing saturated, monounsaturated or polyunsaturated fatty acid (PUFA) residues were subjected to THM-gas chromatography (GC) in the presence of TMAH or TMSH. The obtained results revealed that the THM reaction using TMAH allowed almost quantitative methylation of saturated and monounsaturated fatty acid components independently of the classes of lipids. However, strong alkalinity of TMAH brought about isomerization and/or degradation of PUFA components. In contrast, the use of TMSH was effective to highly sensitive detection of PUFA as well as saturated and monounsaturated fatty acid components contained in triglycerides, phospholipids (phosphatidylcholines) and free fatty acids. On the other hand, TMSH was proved to react hardly with any kind of fatty acid residues in cholesteryl esters due to their steric hindrance.     

Exploring the coordination chemistry of isomerizable terpyridine derivatives for successful analyses of cis and trans isomers by travelling wave ion mobility mass spectrometry

The photochemical cis-trans isomerization of the 4-{4-[2-(pyridin-4-yl)ethenyl]phenyl}-2,2':6',2'-terpyridine ligand (vpytpy) was investigated by UV-vis, NMR and TWIM-MS. Ion mobility mass spectrometry was performed pursuing the quantification of the isomeric composition during photolysis, however an in-source trans-to-cis isomerization process was observed. In order to overcome this inherent phenomenon, the isomerization of the vpytpy species was suppressed by complexation, reacting with iron(ii) ions, and forming the [Fe(vpytpy)(2)](2+) complex. The strategy of "freezing" the cis-trans isomerizable ligand at a given geometric conformation was effective, preventing further isomerization, thus allowing the distinction of each one of the isomers in the photolysed mixture. In addition, the experimental drift times were related to the calculated surface areas of the three possible cis-cis, cis-trans and trans-trans iron(ii) complex isomers. The stabilization of the ligand in a given conformation also allows us to obtain the cis-cis and cis-trans complexes exhibiting the ligand in the metastable cis-conformation, as well as in the thermodynamically stable trans-conformation.     

Cis-trans isomerization of polyunsaturated fatty acid residues in phospholipids catalyzed by thiyl radicals.

Phospholipids containing trans-unsaturated fatty acid residues are the major products of the thiyl radical attack on L-alpha-phosphatidylcholine from soybean lecithin in homogeneous solution or in liposomes (LUVET). Thiyl radicals act as the catalyst for the cis-trans isomerization, and the number of catalytic cycles depends on the reaction conditions. The presence of approximately 0.2 mM oxygen does not influence the reaction outcome but accelerates the efficiency of cis-trans isomerization in homogeneous solution. Under these conditions, the PUFA peroxidation is found to be unimportant. A detailed study of the isomerization of methyl linoleate including product studies indicates the formation of a small amount of conjugated dienes that act as inhibitors. Indeed, all-trans-retinol substantially retarded the isomerization process.     

Synthesis, structural, spectral, and photoswitchable properties of cis- and trans-2,2,2',2'-tetramethyl-1,1'-indanylindanes

As versatile synthetic intermediates for new photoswitchable molecules with a tetramethylindanylindane (stiff-stilbene) core, the cis and trans isomers of 5,16-dibromo-2,2,13',13'-tetramethylindanylindanes 2 were synthesized by the Barton-Kellogg coupling. The bromine atoms of trans-2 could be readily replaced with alkyl (sp3), aryl (sp2), and ethynyl (sp) groups. The cis isomers of the parent tetramethylindanylindane 1 and its bromo derivative 2 were isolated, and their structural and photophysical properties were examined for the first time. Clean and efficient trans-cis and cis-trans photochemical isomerization processes were observed in 1.     

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.     

Reactions of hydrogen atoms with met-enkephalin and related peptides

The reactions of hydrogen atoms with enkephalins and related peptides have been investigated by radiolytic methods in aqueous solutions and lipid vesicle suspensions. Pulse radiolysis experiments indicate that methionine residue (Met) is the main target. In Met-enkephalin (Tyr-Gly-Gly-Phe-Met) the attack of the hydrogen atom occurs to about 50 % on Met with formation of methanethiyl radical. The remaining percentage is divided roughly evenly between Tyr and Phe. With Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) the site of attack is limited to Tyr and Phe. Using a peptide-liposome (that is, 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles) model, the cis-trans isomerization of phospholipids could be detected due to the catalytic action of thiyl radicals. The radiation chemical yields of the H(.) and, consequently of CH(3)S(.) radical, was modulated by the experimental conditions and the nature of peptide. Large amounts of trans lipids observed in phosphate buffer vesicle suspensions indicated the efficient role of double-bond isomerization as marker of Met-containing peptide damage.     

Gas-liquid chromatographic determination of fatty acid composition of cholesteryl esters in human serum using silica Sep-Pak cartridges

A simple and fast analytical procedure for separation and purification of cholesteryl esters of human serum is described. A single lipid extract, together with spiked cholesteryl pentadecanoate, as an internal standard, was passed through a Silica Sep-Pak cartridge. 1.5% diethyl ester in light petroleum was used to elute cholesteryl esters from the column. The separation was verified with thin-layer chromatography on silica gel using light petroleum-diethyl ether-glacial acetic acid (80:20:1) as a solvent. A very clean thin-layer chromatogram of cholesteryl esters without any additional spots of other lipids was obtained. The cholesteryl esters were quantitated by analyzing their fatty acid composition as methyl esters by gas-liquid chromatography. The coefficients of variation were 0.8--4.9% for the major fatty acids (C16:0, C16:1, C18:1, C18:2, C20:4) and 6.7--30.8% for the minor fatty acids (C18:0 and C20:0). The recoveries for cholesteryl palmitate, cholesteryl oleate and cholesteryl linoleate were 90.7, 92.3 and 91.0%, respectively.     

Improved identification of conjugated linoleic acid isomers using silver-ion HPLC separations

Silver-ion high-performance liquid chromatography (Ag+-HPLC) has been shown to be effective in the resolution of most of the isomers of conjugated octadecadienoic acids (18:2), also known as conjugated linoleic acid (CLA). The CLA isomers identified in natural fats from ruminants are a mixture of numerous positional and geometric isomers from 7,9- to 12,14-18:2. Ag+-HPLC separates both geometric (trans,trans < cis/trans < cis,cis) and positional CLA isomers using the mobile phase hexane/acetonitrile (99.9:0.1). The elution volumes for the CLA isomers were not only affected by the concentration of acetonitrile (in the prepared mobile phase) but also with successive runs during the day using a prepared mobile phase batch, due to the partial solubility of acetonitrile in hexane. However, this drift does not affect the relative resolution of the CLA isomers. The addition of diethyl ether to the mobile phase partly stabilizes the solvent mixture. In order to facilitate the interpretation of Ag-+HPLC chromatograms, the relative retention volumes (RRV) were calculated for each CLA isomer. Toluene was added to all the test portions and served as an estimator of dead volume, whereas the elution of the ubiquitous 9c,11t-CLA isomer was chosen as unity (1.00). Expressing the elution of all the CLA isomers as their RRV greatly helped to standardize each CLA isomer, resulting in relatively small coefficients of variation (% CV) for the trans,trans (<1.5%) and cis/trans (<0.5%) CLA isomers. The identification of the CLA isomers was further facilitated by synthesis of authentic CLA isomers. All the geometric CLA fatty acid methyl esters (FAME) from positions 6,8- to 13,15-CLA were commercially available or synthesized by a combination of partial hydrazine reduction of known polyunsaturated fatty acids followed by alkali isomerization, isolation of products, and further iodine-catalyzed geometric isomerization. Based on expressing the elution volume as RRV and the availability of the synthetic CLA isomers, a unique reversal of the elution order of the c/t CLA isomers was found. It is also proposed that the retention times of CLA isomers by gas chromatography (GC) should be expressed as their relative retention times (RRT) relative to methyl gamma-linoleneate. The availability of CLA reference materials and the application of RRV and RRT to Ag+-HPLC and GC separations, respectively, will greatly improve in the identifications of CLA isomers.     

Conformational preference and cis-trans isomerization of 4(R)-substituted proline residues

We report here the conformational preference and prolyl cis-trans isomerization of 4(R)-substituted proline dipeptides, N-acetyl-N'-methylamides of 4(R)-hydroxy-L-proline and 4(R)-fluoro-L-proline (Ac-Hyp-NHMe and Ac-Flp-NHMe, respectively), studied at the HF/6-31+G(d), B3LYP/6-31+G(d), and B3LYP/6-311++G(d,p) levels of theory. The 4(R)-substitution by electron-withdrawing groups did not result in significant changes in backbone torsion angles as well as endocyclic torsion angles of the prolyl ring. However, the small changes in backbone torsion angles phi and psi and the decrease of bond lengths r(Cbeta-Cgamma) or r(Cgamma-Cdelta) appear to induce the increase of the relative stability of the trans up-puckered conformation and to alter the relative stabilities of transition states for prolyl cis-trans isomerization. Solvation free energies of local minima and transition states in chloroform and water were calculated using the conductor-like polarizable continuum model at the HF/6-31+G(d) level of theory. The population of trans up-puckered conformations increases in the order Ac-Pro-NHMe < Ac-Hyp-NHMe < Ac-Flp-NHMe in chloroform and water. The increase in population for trans up-puckered conformations in solution is attributed to the increase in population for the polyproline-II-like conformations with up puckering. The barriers DeltaGct++ to prolyl cis-to-trans isomerization for Ac-Hyp-NHMe and Ac-Flp-NHMe increase as the solvent polarity increases, as seen for Ac-Pro-NHMe. In particular, it was identified that the cis-trans isomerization proceeds through the clockwise rotation about the prolyl peptide bond for Ac-Hyp-NHMe and Ac-Flp-NHMe in chloroform and water, as seen for Ac-Pro-NHMe.