Hoppe-Seyler's Z. Physiol. Chem. Bd. 356, S. 1011 - 1025, Juni 1975
Sterically Hindered Disulfide Bridges in Cystine Diketopiperazine, Cysteinyl-Cysteine Disulfide and Derivatives Michael Ottnad, Peter Hartter, and Günther Jung
(Received 18 December 1974) Dedicated to Prof. Dr. Dr. Günther Weitzel on the occasion of his 60th birthday Summary: L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide · HC1 (2), L-cysteinylcysteine disulfide methyl ester- HC1 (3), and f-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13 C NMR signals of 1 reveals an exceptionally high energy barrier of AG* = 15.8 ± 0.2 kcal/ mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethylsulfoxide at 25 °C, with 80 - 85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller tempera-
ture coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90°, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.
Sterisch gehinderte Disulfid-Brücken in Cystindiketopiperazin, undt-Butyloxycarbonyl-cysteinyl-cystein-disulfid-methylester Zusammenfassung: L-Cystindiketopiperazin (1), L-Cysteinyl-cystein-disulfid * HC1 (2), L-Cysteinyl-cystein-disulfid-methylester· HC1 (3) und f-Butyloxycarbonyl-L-cysteinyl-cystein-disulfid-methylester (4) wurden mit dem CD, der Ultraviolett-, 13 C-NMR-, Infrarot- und Laser-Raman-Spektroskopie untersucht. Aus der Temperaturabhängigkeit der 13C-NMR-Signale wird die außergewöhn-
Cysteinyl-cystein-disulfid-methylester
lich hohe Energiebarriere von 15.8 ± 0.2 kcal/mol für die reversible Helizitatsänderung der inhärent dissymmetrischen Disulfidbrücke von l ermittelt. Das P-helikale Diastereomere liegt in Dirnethylsulfoxid bei 25 °C zu 80 bis 85% vor. Die Cotton-Effekte von l sind intensiver und zeigen geringere Temperatur-Koeffizienten als die konformativ beweglicheren Cystinver-
Address: Prof. Dr. Günther Jung, Chemisches Institut der Universität Tübingen, D-7400 Tübingen, Auf der Morgenstelle. Abbreviations: CD = circular dichroism; NMR = nuclear magnetic resonance; ORD = optical rotatory dispersion; Me2SO = dimethylsulfoxide; Me4Si = tetramethylsilane.
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Bd. 356 (1975)
bindungen 2 und 3. Nach Auflösen der Kristalle von l in 95proz. Äthanol beobachtet man eine zeitabhängige Abnahme der Elliptizität des negativen Cotton-Effekts bei 225 nm, was auf eine Konformationsänderung beim Übergang vom Kristall zur Lösung hindeutet. Neben l sind 2 und 3 zur Zeit die einzigen bekannten Beispiele für Cystinderivate mit C-S-S-C Torsionswinkeln
um 90°, die keine optische Aktivität im Bereich der langwelligen Disulfidabsorption zeigen, so wie es für l das Linderberg-Michl Konzept voraussagt. Bei 305 nm wurde bei l ein bisher nicht beobachteter Cotton-Effekt entdeckt. Die Lösungsmittelabhängigkeit der CD-Spektren von l bis 3 wird diskutiert und die Banden der Infrarot- und Laser-Raman-Spektren werden zugeordnet.
Investigations of cystine and its derivatives and cystinyl peptides by circular dichroism and optical rotatory dispersion have shown that these chiroptical methods are particularly useful for studies of the conformation and conformational changes of disulfide bridges. However, our knowledge of the relations between stereochemistry and the circular dichroism of disulfides is still incomplete. Most of the reported CD studies have been performed on relatively rigid cyclic disulfidesl1'12!. Investigations on non-cyclic disulfides, in particular on L-cystine and its derivatives15'13"221 ( have shown that no definite conclusions can be based on the CD spectra because of the flexibility of the residues on the disulfide group and the rotation of the C-S-S-C grouping.
previous CD work on 5-alkylthio-L-cysteines[211 we could show that the influence of the alkyl group depends on the nature of the alkyl residue. However, this effect is rather small and results mainly in a shift of the maximum of the long wavelength disulfide Cotton effect. Torsional angles and helicity of these compounds must be very similar to those of cystine. The temperature and solvent dependence of the CD spectrum of oxidized glutathione revealed a solvent-dependent interaction of the disulfide group with the amide groups, resulting in a bathochromic shift of these chromophoresf221. The temperature dependence of the CD spectrum of G-SS-G can be explained by rotations on the C-S-S-C group. This finding is supported by a 13C NMR study, which revealed a distinct non-linear temperature dependence of the cystine Ca and C^ signals130!. Raman spectra of G-SS-G in solid state and solution showed that the torsional angle of the disulfide group does not change upon dissolution'301. This is definitely not the case for all cystinyl peptides.
The electronic structure of the disulfide group allows the formation of two enantiomers with P- and M-helicity'231, which have the same energy content. The presence of a further center of chirality gives diastereomers, which have, in the case of L-cystine, the configuration/? on Ca and P- or M-helical disulfide arrangements. Because of the small differences in the thermodynamic stability of these diastereomers^211 and the relatively low rotational barriers of the C-S-S-C group [24-27]^ a mixture is found in solution. The relative distribution of the diastereomers depends on the temperature, as shown by the large temperature dependence of the ORD^281 and CD spectra of cystinel151 and N,N'-diacetyl-L-cystine-bis-methylamide1181. Also, in the solid state, there is a preference for one of the two possible diastereomers. For example, two disulfide groups in lysozyme possess P-, and the other two M-helicityi291. Only few examples of CD investigations on smaller cystine peptides with definite assignments of the CD bands have been reported so far. In
In recent experiments, we detected a strong time dependence of the CD spectra of some cystine peptides, e.g. Boc-Cys-Gly-Cys-OMe, upon dissolution in ethanoli311, which is due to conformational changes from the crystalline to the dissolved state. Most of the authors of previously published CD studies did not consider this possibility, which may be one reason for the poor reproducibility of some published data of molar ellipticities. In the following we report CD and 13C NMR studies on cystine dipeptides, which should exhibit structural and chiroptical similarities between the spectra of simple, low molecular weight and non-peptidic model compounds and those of oligopeptides containing cystine. In subsequent papers we will report on CD studies of various
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cystinyl peptides Cys-X„-Cys*. This logical approach should fill to some extent the gap in the present knowledge of the dynamic stereochemistry of cystine peptides, which extends from the oligopeptide region up to the well investigated oxytocines. The diketopiperazine of L-cystine 1 ("Cyclo-Lcystine"i?J or 3,4-dithia-7,9-diazabicyclo(4.2.2> decan-8,10-dione)has received considerable attention as an experimental model for the proof of theoretical considerations of the chiroptical properties of disulfides'32"34^. The compound has a bent diketopiperazine ring bridged by a disulfide linkage with a torsional angle of 0 = 90°l71. According to the theory, none of the molecules with such a disulfide grouping should exhibit optical activity in the long wavelength disulfide absorption around 245 nm. In fact, this was experimentally observed in a CD spectrum of l'7'. Our interest in the dynamic stereochemistry of cystine peptides prompted us to investigate the temperature-induced conformational changes in this relatively rigid molecule, which may occur by rotations around the disulfide group corresponding to a reversible conversion of the diastereomer Ib with M-helicity into diastereomer la with P-helicity (Fig. 1).
la: P-helical
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Ib: M-helical
Fig. 1. Diastereomers of cystine diketopiperazine with M and P-helical arrangement of the C-S-S-C grouping! 71.
According to 1 H NMR spectra, the isomer la predominates in Me2SO solution171. A study of the temperature dependence of the 13C NMR should allow a quantitative determination of the energy required for this interconversion. Some results of * Ottnad, M., Hartter, P. & Jung, G., unpublished results.
these studies have recently been published by us in a short note1351. In addition, the solvent and temperature dependence of the CD should indicate the origin of the Cotton effects and the role of solvent stabilization on both forms. Finally, infrared and laser Raman spectra should indicate the structural features of 1 in the solid state. It is expected that 1 prefers in the crystalline state either P- or M-helicity of the disulfide bridge, whereas in solution an equilibrium exists between the two forms (Fig. 1). Because of the rigidity of the molecule, one might detect time-dependent phenomena by recording spectra immediately after dissolving crystals of 1. ®H2-Cys-Cys-OH, Cle 2 e
e
H2-Cys-Cys-OMe, Cl
Boc-Cys-Cys-OMe
Conformational calculations'36' on the heterode tic cyclic L-cysteinyl-cysteine disulfide 2 have shown that this compound possesses a considerable conformational flexibility compared to 1. However, the torsional angle of the disulfide group is also expected to be around 90°. Therefore, in addition to 1, the heterodetic cyclic dipeptides L-cysteinyl-cysteine disulfide (2), its methyl ester (3) and r-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) seem to be promising tools for the investigation of chiroptical properties of disulfides. In the following, we report a comparative conformational study of the rigid cystine diketopiperazine 1 and the more flexible cysteinyl-cysteine disulfide derivatives 2 - 4. Materials and Methods Compounds and Solvents L-Cystine diketopiperazine, L-cysteinyl-cysteine disulfide and its methyl ester and f-butyloxycarbonyl-Lcysteinyl-cysteine disulfide methyl ester are prepared as described*37'38!. All compounds are highly purified and crystalline. L-Cystine diketopiperazine is obtained from ethanol/CHCl3 in very thin needles up to 3.5 cm long. All solvents used for recrystallization prior to the spectroscopic measurements are p.a. grade. [2H6]Me2SO and
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all spectral grade solvents for the CD measurements are from E. Merck AG, Darmstadt. Methods The CD spectra were recorded on a Roussel-Dichrograph II. The temperature of the cell was controlled by a Haake thermostat model FK 2 within ±0.3 °C. All spectra were recorded at least twice using cells of different path lengths (0.1 to 20 mm). The reproducibility was checked within each series to exclude the recording of possible decomposition products. The ultraviolet spectra of the same solutions were recorded on a Gary 15 ultraviolet spectrometer. For the infrared measurements, we used a Perkin-Elmer instrument, and the Raman spectra were recorded on the Jeol JRS-1 Laser Raman Spectrometer. The temperature dependence of the 13C NMR signals was recorded on a Bruker HFX-90-PFT instrument with an 18-inch magnet (ft = 22.628 MHz for 13C,/2 = 90 MHz for 1H, /0 = 13 MHz for 2H), using ^-broadband decoupling and 2 H stabilization. [ 2 H6)Me 2 SO was used as solvent and 2H-lock in 10-mm cells. A concen-
Bd. 356(1975)
tration of 200 mg dipeptide per 1.5 ml [ 2 H 6 ]Me 2 SO was used. Each spectrum of L-cystine diketopiperazine required the accumulation of at least 4 096 pulse interferograms (pulse widths 4 jusec, pulse interval 0.4 sec, sweep 200 Hz/cm) in a Fabritek-1074 Computer (4k). After Fourier-transformation in a PDP-8-I computer (4k), the phase-corrected real-part spectra were recorded. The channel differences of the signals with respect to the [ 2 H6lMe 2 SO were converted into ppm values relative to TMS ext = 0 ppm. The error is not more than 0.1 ppm.
Results and Discussion Temperature dependence of the Cotton ofcystine diketopiperazine
effects
In contrast to the CD of open-chain disulfides, and in agreement with the theory, no Cotton effect is found for 1 in the long wavelength disulfide ultraviolet absorption band. Fig.2 shows only two intense Cotton effects at 222 nm (—)
Fig. 2. Solvent dependence of the circular dichroism spectra of cystine diketopiperazine.
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and 194 nm (+). The negative Cotton effect corresponds to a η-π* transition of the two cis amide groups. And according to the octant rule for carbonyl compounds, a P-helical arrangement of the disulfide grouping is very likely in solution^7'. The Cotton effect at 194 nm is a superposition of the π — π* amide transition and a disulfide transition. Compared to the CD spectrum of oxidized L-glutathione117·21'22'391, the molar ellipticities of both transitions of 1 have opposite signs and are much higher. This is explained by the conformational rigidity of 1 and the geometric position of the cis amide groups, which favors strong electric dipole dipole interactions.
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r
As expected, the temperature dependence of the CD of 1 shows a curve characteristic of the conform ational dynamics of diastereomers. The temperature coefficients of all Cotton effects of 1 are smaller than those for other cystine compounds [16-19,21,22,39] and ^ structurally related djenkolic acid^40', which has also an inherent dis100 20 -10 0 iO symmetric chromophore. The absence of the 245-nm disulfide Cotton effect allows no direct Fig. 3. Temperature dependence of the Cotton effect of observation of the interconversion of the P-helical cystine diketopiperazine at 225 nm in O.lN HC1 diastereomer into the M-helical isomer and vice (o o), in trifluoroethanol (a a), and in versa. However, the amide transitions should also ethanol (· ·). depend largely on these temperature-induced trifluoroethanol, the bends observed in the teminterconversions. Indeed, a rise in temperature perature dependence differ by more than 10 °C from 0 to 55 °C reduces the molar ellipticities at (Fig.3). This fact, and the observation of larger 222 nm to about 38% and at 194 nm to 26%. ellipticities of 1 in trifluoroethanol, indicate that Fig. 3 shows part of the sigmoid curve obtained this more strongly hydrogen-bonding solvent by plotting the standardized ratios of the molar stabilizes one of the two diastereomers. The dioxellipticities [ ]t/[ ]55 versus the corresponding ane, ethanol and methanol solutions show temperature t. smaller molar ellipticities for both Cotton efBelow 40 °C the thermodynamically more stabile fects. As expected, the η - π* Cotton effect of P-helical diastereomer predominates, and with theamide groups shifts hypsochromically with rising temperature, the population of the conincreasing solvent polarity from 231 nm in dioxformation with an M-helical disulfide group, prob- ane to 222 nm in trifluoroethanol (Table 1). A ably exhibiting a less negative Cotton effect, inhypsochromic shift is also observed for the posicreases. Above 40 °C there is only a minor detive short-wavelength Cotton effect. A weak crease in the ellipticity ratio due to the balanced Cotton effect was discovered at 305 nm in mepopulation of the two conformations. thanol and dioxane solutions of 1 (Fig. 2). Its ellipticity and position have only minor temperSolvent dependence of the CD of cystine ature and solvent dependence. Since different diketopiperazine batches of highly purified crystals of 1 have been A linear decrease in ellipticity of the η — τι* used in several independent measurements, the Cotton effect is observed in 0.1 N HC1 between origin of this new Cotton effect is certainly not 20 and 100 °C (Fig.3). In ethanol (100%) and in due to an impurity. It is possible that a positive
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1016 Table 1. Position and molar ellipticities of the Cotton effects of cyclo-L-cystine.
M. Ottnad, P. Hartter and G. Jung
Solvent
Temp. [°C]
Dioxane
20
Methanol
20
Ethanol(95%)
25
0.1NHC1 Trifluoroethanol
20 20
Cotton effect above 300 nm arising from a chargetransfer transition is superposed by the negative amide Cotton effect to such an extent that a positive maximum below 300 nm appears. A similar phenomenon is observed in the case of 6,8-thioctic acid1101. The different perturbation of the amide transition in the Ρ and M-helical arrangements induces different CD band contours in the two forms. As a consequence, the CD band of one conformation is superposed by the flatter band of the other form, resulting in the weak positive Cotton effect at 305 nm. This explanation is supported by the symmetric shape of the 305 nm band. Referring to studies with epidithiadiketopiperaznW41', it seems reasonable that a CT transition transfers an electron from an occupied na, nb sulfur orbital into the antibonding orbital of the amide groups of 1. Such a CT transition may be possible because of the close proximity of the sulfur atoms and the carbonyl groups in the P-helical form. Because of the 250 nm disulfide absorption, such a transition is not observed. However, the absence of a solvent dependence contradicts this assumption, so further experiments will be necessary in this context. Time dependence of the CD ofcystine diketopiperazine Because of the relatively high rotational barrier between the conformational isomers la and Ib, it should be possible to detect one of these two forms by measuring freshly prepared solutions of 1, provided the crystal embeds only one form. In the first paper on the conformation and configuration of cyclo-L-cystinel7], it was pointed
Bd. 356 (1975)
λ (nm] 305 231 305 226 197
227,5 198,5 224 222 194
[ o j [degxcm 2 xdmol~ 1 ] 31 51480 16 56 000 327070 51100 259 850 - 52870 - 62130 360 340 -
out that the M-helical form could possibly exhibit a smaller negative (or even slightly positive) rotational strength .R than the P-helical diastereomer. Due to the influence of the -CH2-S- group in a positive octant relative to the peptide carbonyl chromophor, la should possess the higher and negatived when compared to Ib. The spectroscopic registration of an excess of either la or Ib directly after dissolution of the crystals before equilibrium is reached should give information on the screw sense in the crystalline state. However, such a conclusion can be equivocal when the Mhelical form Ib possesses a stronger overall rigidity, including solvent shell influences. Further complications may arise when 1 dissolves in an aggregated state, which generally exhibits stronger Cotton effects than the corresponding monomers. In this case, a decrease in ellipticity will be found as long as the oligomers dissociate into monomers. Table 2 shows the results of a first tentative experiment on the time dependence of the CD after dissolving 1 in 95 % ethanol at 4 °C and 25 °C. Indeed, a systematic change in the intensity with time is found. The decrease in ellipticity is expected for a change from a predominantly P-helical population to aP- and M-helical mixture, not considering possible restrictions. The decrease in ellipticity of the η — π* transition at 227 nm is finished after about three days at 4 °C, corresponding to a total decrease of 15 - 20%. In all previous and common experiments investigating the temperature dependence of the CD of 1, a normal decrease with increasing temperature was found. Because the distribution of the diastereomers comes to an equilibrium faster at 25 °C, the time dependence is less pronounced in
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Table 2. Time-dependence of the CD after dissolving crystals of cystine diketopiperazine.
Time [h]
[Θ] [degx cm2 χ dmol
Solution in 95%ethanol at4°C \ max = 227 nm
0.5 20 46 68
-
Solution in 95%ethanolat25°C
1 21
- 54000 - 53200
this case. However, it is understood that many of the CD data obtained by us and others can be superimposed by a time dependence. A detailed spectroscopic kinetic analysis of these time-dependent phenomena, including an investigation of solvent, temperature and possible concentration effects, will be reported elsewhere. As yet the experiments should by no means be considered as indicative of a partial Ρ -> Μ transition of 1 going from crystal to solution, but as a new and promising technique which has already been very useful in similar cases^31'. Temperature dependence of the 13CNMR of cystine diketopiperazine Several aspects of the exact estimation of energy barriers by pulsed NMR techniques are problematic and require a complete line shape analysis142"441. Since the population of the less stable M-helical conformation Ib is relatively small, a broadening of the line width could not be observed because of the small S/N ratio of the Ib signals (Fig.4, Tab.3). Table 3. Temperature dependence of the
l
]
56250 51950 48 900 47850
However, an improved S/N ratio requires very long accumulation times, which are intolerable, in particular at high temperature. Below 40 °C there are three weak carbon signals besides the three intense signals of the predominating diastereomer, giving proof for the exact C2 symmetry of both forms*71. The stronger signal of the carbonyl carbon is found at lower field. This indicates, independently from the CD, that there is a close proximity between the sulfur atoms and the carbonyl group in the P-helical diastereomer la. The deshielding of the carbonyl group in la could be caused by a through-space electric field effect of the 3p lone pair electrons of the sulfur atoms shifting the signal to lower field than the corresponding signal of the M-helical diastereomer Ib. Large through-space effects of the sulfur of cysteine to its C = 0 group are long known to us from the pH and temperature dependence of the 13C NMR of oxidized and reduced glutathionef30'45'461 and of cysteine and related aminothiols130'471. Solvent shell influences must be taken into account for the ob-
13
C NMR signals of cystine diketopiperazine in [2H6]Me2SO. 13
Temp. [°C] Cys-CO 25 28 35 40 45 50 55 65 75 98
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Sterically Hindered Bisulfide Bridges
Bd. 356(1975)
Ρ
Μ
170.69 170.67 170.67 170.47 170.47 170.47 170.36 170.25 170.14 169.39
166.81 166.67 166.67
C chemical shift δ [ppm]Me4Si = 0 Cys-Ca Μ Ρ 56.32 56.31 56.31 56.31 56.31
53.84 53.84 53.73 53.73 53.84 53.73 53.73 53.73 53.84 54.05
Cys-C0 Ρ
47.58 47.58 47.58 47.47 47.47 47.37 47.37 47.26 47.14 46.71
Μ
44.67 44.67 44.67 44.67 45.64
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z
u
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Sterically Hindered Bisulfide Bridges
served shift differences of the two diastereomers, although these are considered in general to be small. However, we observed common upfleld shifts of 0.6 to 1.5 ppm of C and C = 0 resonances of amino acids in the lipophflic octadecapeptide antibiotic alamethicin in going from chloroform or methanol solution to the strongly polar dimethylsulfoxide'481. Therefore, the observed upfield shifts of the Cys-COand Cys-C^ in cystine diketopiperazine in changing from the P to the M-helical disulfide arrangement may be also due to a small extent to solvent influences. An increased solvent-solute interaction is expected for the M-helical diastereomer Ib, which has more exposed peptide carbonyls. As expected, the conformational inner molecular changes and accompanying solvent influences shift the position of the Cys-Ca to a minor extent. Whereas Cys-C^ and Cys-CO shift to higher field, in the case of C a , a reverse shift to lower field is found for the M-helical form. The somewhat larger shift difference for the peptide carbonyls (Δ = 3.9ppm) with respect to Cys-C^ (Δ = 2.5 ppm) and Cys-C^ (Δ = 2.9 ppm) is not unexpected (Tab.3). It has long been known that sp2 hybridized carbons in general show the largest shift effects on electrostatic changes of their environment'491. The theoretical relationships between electron density and carbon chemical shifts are not very satisfying as yet'49'501. Particularly in the case of the complex dissociation equilibrium of thiols and disulfides, a clear-cut differentiation between the through-space electric field effects expected to produce downfield shifts'511 and the inductive through-bond effects1521 acting in the opposite direction is rather difficult^47'531. However, our experiments so far indicate particularly large through-space influences of the sulfur 3p orbitals on nearby carbons, which often give rise to 13C chemical shifts opposite to those observed in corresponding non-sulfur analogs. In the present case, the optical measurements prove an interaction between the disulfide and carbonyl chromophores which changes the excitation energy of the η-π* transition. It is evident that this perturbation also has a strong influence on the paramagnetic shielding σρ of the carbonyl 13C nuclei. In the case of the diketopiperazine of cystine, a rise in temperature results only in a relatively small shift (Table 3) when compared to the tem-
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perature-induced shifts observed for the cystine carbons in oxidized glutathione'301. Above 50 °C, the smaller signals of the M-helical diastereomer disappear and the interconversion of la and Ib occurs so rapidly that one observes only single signals. These signals shift with further increase of the temperature due to the decrease in viscosity and solvent/solute interactions. This investigation is the first determination of an energy barrier in a peptide and a disuifide using 13 C NMR. Although we are aware of all the advantages of this method and all the objections concerning the reliability of the determination of energy barriers from pulse Fourier-transform 13C NMR spectra, we estimated the activation enthalpy of the interconversion. From a logarithmic plot'421 of Δι;, the difference of the 13C resonances of the two conformers (expressed in Hz), versus Δ G # with T(° K) as parameter, Δ0# values of 15.6 to 16.0 kcal/mol are obtained. In this context, it is interesting that the highest known value of another example of a an energy barrier in a hindered diaryl disulfide was found to be 15.7 kcal/mole'541. As for 1, this can be considered only as the lower limit for these processes. Unfortunately, our measurements are restricted as yet to Me2SO solutions because of the low solubility of 1. From the intensities of the 13C NMR signals of la and Ib, the percentage of the less stable M-helical form Ib can be estimated to be between 15 and 20% at 25 °C. This estimation is reliable, since the relaxation times and the nuclear Overhauser enhancements of the aliphatic carbons in both forms should be comparable. Measurements of the longitudinal relaxation times Τ ι well above the coalescence point, about 60 °C, and below, about 0 °C, should reveal further evidence of the intramolecular motion of 1. Circular dichroism of cysteinyl-cysteine disulfide and derivatives The hydrolysis of one of the two cis amide bands in the homodetic and heterodetic, bicyclic cystine diketopiperazine 1 yields the heterodetic monocyclic cysteinyl-cy steine disulfide 2. Conformational calculations for 2 predict a non-planar cis amide bond and bond lengths and angles similar to those of open chain cystine peptides. The con-
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M. Ottnad, P. Hartter and G. Jung
formational energies of the P and M-helical forms of 2 should differ only by about 0.8 kcal/mole[361. Therefore a CD spectrum with a relatively weak Cotton effect around 250 to 265 run, exhibiting a temperature dependence similar to that of glutathione^22' or cystine, is expected. In contrast to this expectation, we observe a spectrum similar to that of 1, except for the opposite signs and lower eilipticities of the Cotton effects (Fig. 5). The differences are due to the fact that the single amide bond in 2 has no dipole-dipole interactions with a second one, as is the case for 1. The reduced eilipticities are partly due to the higher flexibility of 2. The solvent dependence of the CD of 2 shows that the positive Cotton effect at 225 to 230 run originates from an amide transition, and the short wavelength CD band has the same origin as in 1 (Tab.4). The long wavelength,
Bd. 356 (1975)
negative and weak Cotton effect is due to a disulfide transition, because it shows a large temperature dependence in methanol and in 0.1 N HC1, and also a bathochromic shift with increasing temperature. The temperature-induced decrease in the eilipticities at 200 and 227 nm is relatively small. The weak negative maximum of the longest wavelength Cotton effect may be due to an overlapping of a disulfide transition with the strong positive amide CD band. Therefore a position at much longer wavelength is found when compared to glutathione or cystine, and also the bathochromic shift of this maximum with increasing temperature is readily explained. The CD spectra of 3 are almost identical with those of 2 (Fig. 5) in the short-wavelength region. However, due to the presence of the ester group, there are differences in the long wavelength region. As with 1, there are no long-wavelength
Fig. 5. Circular dichroism spectra of f-butyloxycarbonyl-L-cysteinyl-cysteine-disulfide methyl ester (4) ( ), L-cysteinyl-cysteinsdisulfide methyl ester hydrochloride (3) ( ) and L-cysteinyl-cysteine-disulfide hydrochloride (2) ( ) at 20 °C in absolute ethanol.
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Bd. 356 (1975)
Sterically Hindered Bisulfide Bridges
Cotton effects in strongly polar solvents, e.g., O.lN HC1, trifluoroethanol and hexafluoroacetone trihydrate. The negative Cotton effect at 285 nm found in methanol shows the same temperature dependence as the corresponding band of 2, an apparent CD maximum of a disulfide transition overlapping with the strong and positive amide band. This is confirmed by the shoulder found in the ultraviolet spectrum of 3 at 245 nm corresponding to the absorption range of disulfides with torsional angles around 90°. The CD spectrum of 3 in absolute ethanol shows a small positive Cotton effect at 305 nm that has also been found for cystine diethyl ester in propanol solution. It is not possible to predict the helicity of the disulfide groups of 2 and 3 from the CD, since the ellipticities of the Cotton effects are too small, and they may be largely influenced by effects of their environment. The introduction of the bulky f-butyloxycarbonyl group gives the fully protected dipeptide 4, which exhibits a CD spectrum in absolute ethanol (Fig. 5) deviating considerably from those of 2 and 3. The two long wavelength Cotton effects at 260 nm (+) and 243 nm (—) are due to a disulfide transition. They are situated symmetrically to 257 nm. This may be due to a torsional angle similar to that of open-chain disulfides. These assignments of the long-wavelength CD bands are supported by the finding that N,N'-dimethyl-L-cystine also exhibits two CD bands at 280 nm (+) and 247 nm (-) in 0.1 N HC1. Therefore the successive introduction of substituents on the amino group seems to induce a common effect on the CD of cystine derivatives. This band splitting is also observed in chiraldialkyldisulfidesl5]. The change from methanol or ethanol to the less polar solvent dioxane reduces the ellipticities of the long-wavelength bands drastically and gives a bathochromic shift of 40 nm. On the other hand, the ellipticity of the amide band is severalfold increased. This effect is also observed on the heterodetic monocyclic tripeptide disulfides Boc-Cys-X-Cys-OMe [X =[38 Gly, Ala, Leu, Phe, Val,Glu(OBuO,Lys(Z)] K * Ottnad, M., Hartter.P. & Jung, G., unpublished results.
1021
"ο
ε
^Η ο Ό V)OO^> Ο Ο Ο Ο
χ
°ε χ 1
r-.^- -H rs co oo
r- —ι
,-
Sterically Hindered Disulfide Bridges in Cystine Diketopiperazine, Cysteinyl-Cysteine Disulfide and Derivatives Michael Ottnad, Peter Hartter, and Günther Jung
(Received 18 December 1974) Dedicated to Prof. Dr. Dr. Günther Weitzel on the occasion of his 60th birthday Summary: L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide · HC1 (2), L-cysteinylcysteine disulfide methyl ester- HC1 (3), and f-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13 C NMR signals of 1 reveals an exceptionally high energy barrier of AG* = 15.8 ± 0.2 kcal/ mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethylsulfoxide at 25 °C, with 80 - 85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller tempera-
ture coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90°, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.
Sterisch gehinderte Disulfid-Brücken in Cystindiketopiperazin, undt-Butyloxycarbonyl-cysteinyl-cystein-disulfid-methylester Zusammenfassung: L-Cystindiketopiperazin (1), L-Cysteinyl-cystein-disulfid * HC1 (2), L-Cysteinyl-cystein-disulfid-methylester· HC1 (3) und f-Butyloxycarbonyl-L-cysteinyl-cystein-disulfid-methylester (4) wurden mit dem CD, der Ultraviolett-, 13 C-NMR-, Infrarot- und Laser-Raman-Spektroskopie untersucht. Aus der Temperaturabhängigkeit der 13C-NMR-Signale wird die außergewöhn-
Cysteinyl-cystein-disulfid-methylester
lich hohe Energiebarriere von 15.8 ± 0.2 kcal/mol für die reversible Helizitatsänderung der inhärent dissymmetrischen Disulfidbrücke von l ermittelt. Das P-helikale Diastereomere liegt in Dirnethylsulfoxid bei 25 °C zu 80 bis 85% vor. Die Cotton-Effekte von l sind intensiver und zeigen geringere Temperatur-Koeffizienten als die konformativ beweglicheren Cystinver-
Address: Prof. Dr. Günther Jung, Chemisches Institut der Universität Tübingen, D-7400 Tübingen, Auf der Morgenstelle. Abbreviations: CD = circular dichroism; NMR = nuclear magnetic resonance; ORD = optical rotatory dispersion; Me2SO = dimethylsulfoxide; Me4Si = tetramethylsilane.
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1012
M. Ottnad, P. Hartter and G. Jung
Bd. 356 (1975)
bindungen 2 und 3. Nach Auflösen der Kristalle von l in 95proz. Äthanol beobachtet man eine zeitabhängige Abnahme der Elliptizität des negativen Cotton-Effekts bei 225 nm, was auf eine Konformationsänderung beim Übergang vom Kristall zur Lösung hindeutet. Neben l sind 2 und 3 zur Zeit die einzigen bekannten Beispiele für Cystinderivate mit C-S-S-C Torsionswinkeln
um 90°, die keine optische Aktivität im Bereich der langwelligen Disulfidabsorption zeigen, so wie es für l das Linderberg-Michl Konzept voraussagt. Bei 305 nm wurde bei l ein bisher nicht beobachteter Cotton-Effekt entdeckt. Die Lösungsmittelabhängigkeit der CD-Spektren von l bis 3 wird diskutiert und die Banden der Infrarot- und Laser-Raman-Spektren werden zugeordnet.
Investigations of cystine and its derivatives and cystinyl peptides by circular dichroism and optical rotatory dispersion have shown that these chiroptical methods are particularly useful for studies of the conformation and conformational changes of disulfide bridges. However, our knowledge of the relations between stereochemistry and the circular dichroism of disulfides is still incomplete. Most of the reported CD studies have been performed on relatively rigid cyclic disulfidesl1'12!. Investigations on non-cyclic disulfides, in particular on L-cystine and its derivatives15'13"221 ( have shown that no definite conclusions can be based on the CD spectra because of the flexibility of the residues on the disulfide group and the rotation of the C-S-S-C grouping.
previous CD work on 5-alkylthio-L-cysteines[211 we could show that the influence of the alkyl group depends on the nature of the alkyl residue. However, this effect is rather small and results mainly in a shift of the maximum of the long wavelength disulfide Cotton effect. Torsional angles and helicity of these compounds must be very similar to those of cystine. The temperature and solvent dependence of the CD spectrum of oxidized glutathione revealed a solvent-dependent interaction of the disulfide group with the amide groups, resulting in a bathochromic shift of these chromophoresf221. The temperature dependence of the CD spectrum of G-SS-G can be explained by rotations on the C-S-S-C group. This finding is supported by a 13C NMR study, which revealed a distinct non-linear temperature dependence of the cystine Ca and C^ signals130!. Raman spectra of G-SS-G in solid state and solution showed that the torsional angle of the disulfide group does not change upon dissolution'301. This is definitely not the case for all cystinyl peptides.
The electronic structure of the disulfide group allows the formation of two enantiomers with P- and M-helicity'231, which have the same energy content. The presence of a further center of chirality gives diastereomers, which have, in the case of L-cystine, the configuration/? on Ca and P- or M-helical disulfide arrangements. Because of the small differences in the thermodynamic stability of these diastereomers^211 and the relatively low rotational barriers of the C-S-S-C group [24-27]^ a mixture is found in solution. The relative distribution of the diastereomers depends on the temperature, as shown by the large temperature dependence of the ORD^281 and CD spectra of cystinel151 and N,N'-diacetyl-L-cystine-bis-methylamide1181. Also, in the solid state, there is a preference for one of the two possible diastereomers. For example, two disulfide groups in lysozyme possess P-, and the other two M-helicityi291. Only few examples of CD investigations on smaller cystine peptides with definite assignments of the CD bands have been reported so far. In
In recent experiments, we detected a strong time dependence of the CD spectra of some cystine peptides, e.g. Boc-Cys-Gly-Cys-OMe, upon dissolution in ethanoli311, which is due to conformational changes from the crystalline to the dissolved state. Most of the authors of previously published CD studies did not consider this possibility, which may be one reason for the poor reproducibility of some published data of molar ellipticities. In the following we report CD and 13C NMR studies on cystine dipeptides, which should exhibit structural and chiroptical similarities between the spectra of simple, low molecular weight and non-peptidic model compounds and those of oligopeptides containing cystine. In subsequent papers we will report on CD studies of various
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Bd. 356 (1975)
cystinyl peptides Cys-X„-Cys*. This logical approach should fill to some extent the gap in the present knowledge of the dynamic stereochemistry of cystine peptides, which extends from the oligopeptide region up to the well investigated oxytocines. The diketopiperazine of L-cystine 1 ("Cyclo-Lcystine"i?J or 3,4-dithia-7,9-diazabicyclo(4.2.2> decan-8,10-dione)has received considerable attention as an experimental model for the proof of theoretical considerations of the chiroptical properties of disulfides'32"34^. The compound has a bent diketopiperazine ring bridged by a disulfide linkage with a torsional angle of 0 = 90°l71. According to the theory, none of the molecules with such a disulfide grouping should exhibit optical activity in the long wavelength disulfide absorption around 245 nm. In fact, this was experimentally observed in a CD spectrum of l'7'. Our interest in the dynamic stereochemistry of cystine peptides prompted us to investigate the temperature-induced conformational changes in this relatively rigid molecule, which may occur by rotations around the disulfide group corresponding to a reversible conversion of the diastereomer Ib with M-helicity into diastereomer la with P-helicity (Fig. 1).
la: P-helical
1013
Sterically Hindered Disulfide Bridges
Ib: M-helical
Fig. 1. Diastereomers of cystine diketopiperazine with M and P-helical arrangement of the C-S-S-C grouping! 71.
According to 1 H NMR spectra, the isomer la predominates in Me2SO solution171. A study of the temperature dependence of the 13C NMR should allow a quantitative determination of the energy required for this interconversion. Some results of * Ottnad, M., Hartter, P. & Jung, G., unpublished results.
these studies have recently been published by us in a short note1351. In addition, the solvent and temperature dependence of the CD should indicate the origin of the Cotton effects and the role of solvent stabilization on both forms. Finally, infrared and laser Raman spectra should indicate the structural features of 1 in the solid state. It is expected that 1 prefers in the crystalline state either P- or M-helicity of the disulfide bridge, whereas in solution an equilibrium exists between the two forms (Fig. 1). Because of the rigidity of the molecule, one might detect time-dependent phenomena by recording spectra immediately after dissolving crystals of 1. ®H2-Cys-Cys-OH, Cle 2 e
e
H2-Cys-Cys-OMe, Cl
Boc-Cys-Cys-OMe
Conformational calculations'36' on the heterode tic cyclic L-cysteinyl-cysteine disulfide 2 have shown that this compound possesses a considerable conformational flexibility compared to 1. However, the torsional angle of the disulfide group is also expected to be around 90°. Therefore, in addition to 1, the heterodetic cyclic dipeptides L-cysteinyl-cysteine disulfide (2), its methyl ester (3) and r-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) seem to be promising tools for the investigation of chiroptical properties of disulfides. In the following, we report a comparative conformational study of the rigid cystine diketopiperazine 1 and the more flexible cysteinyl-cysteine disulfide derivatives 2 - 4. Materials and Methods Compounds and Solvents L-Cystine diketopiperazine, L-cysteinyl-cysteine disulfide and its methyl ester and f-butyloxycarbonyl-Lcysteinyl-cysteine disulfide methyl ester are prepared as described*37'38!. All compounds are highly purified and crystalline. L-Cystine diketopiperazine is obtained from ethanol/CHCl3 in very thin needles up to 3.5 cm long. All solvents used for recrystallization prior to the spectroscopic measurements are p.a. grade. [2H6]Me2SO and
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1014
M. Ottnad, P. Hartter and G. Jung
all spectral grade solvents for the CD measurements are from E. Merck AG, Darmstadt. Methods The CD spectra were recorded on a Roussel-Dichrograph II. The temperature of the cell was controlled by a Haake thermostat model FK 2 within ±0.3 °C. All spectra were recorded at least twice using cells of different path lengths (0.1 to 20 mm). The reproducibility was checked within each series to exclude the recording of possible decomposition products. The ultraviolet spectra of the same solutions were recorded on a Gary 15 ultraviolet spectrometer. For the infrared measurements, we used a Perkin-Elmer instrument, and the Raman spectra were recorded on the Jeol JRS-1 Laser Raman Spectrometer. The temperature dependence of the 13C NMR signals was recorded on a Bruker HFX-90-PFT instrument with an 18-inch magnet (ft = 22.628 MHz for 13C,/2 = 90 MHz for 1H, /0 = 13 MHz for 2H), using ^-broadband decoupling and 2 H stabilization. [ 2 H6)Me 2 SO was used as solvent and 2H-lock in 10-mm cells. A concen-
Bd. 356(1975)
tration of 200 mg dipeptide per 1.5 ml [ 2 H 6 ]Me 2 SO was used. Each spectrum of L-cystine diketopiperazine required the accumulation of at least 4 096 pulse interferograms (pulse widths 4 jusec, pulse interval 0.4 sec, sweep 200 Hz/cm) in a Fabritek-1074 Computer (4k). After Fourier-transformation in a PDP-8-I computer (4k), the phase-corrected real-part spectra were recorded. The channel differences of the signals with respect to the [ 2 H6lMe 2 SO were converted into ppm values relative to TMS ext = 0 ppm. The error is not more than 0.1 ppm.
Results and Discussion Temperature dependence of the Cotton ofcystine diketopiperazine
effects
In contrast to the CD of open-chain disulfides, and in agreement with the theory, no Cotton effect is found for 1 in the long wavelength disulfide ultraviolet absorption band. Fig.2 shows only two intense Cotton effects at 222 nm (—)
Fig. 2. Solvent dependence of the circular dichroism spectra of cystine diketopiperazine.
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Bd. 356 (1975)
Sterically Hindered Disulfide Bridges
and 194 nm (+). The negative Cotton effect corresponds to a η-π* transition of the two cis amide groups. And according to the octant rule for carbonyl compounds, a P-helical arrangement of the disulfide grouping is very likely in solution^7'. The Cotton effect at 194 nm is a superposition of the π — π* amide transition and a disulfide transition. Compared to the CD spectrum of oxidized L-glutathione117·21'22'391, the molar ellipticities of both transitions of 1 have opposite signs and are much higher. This is explained by the conformational rigidity of 1 and the geometric position of the cis amide groups, which favors strong electric dipole dipole interactions.
1015
r
As expected, the temperature dependence of the CD of 1 shows a curve characteristic of the conform ational dynamics of diastereomers. The temperature coefficients of all Cotton effects of 1 are smaller than those for other cystine compounds [16-19,21,22,39] and ^ structurally related djenkolic acid^40', which has also an inherent dis100 20 -10 0 iO symmetric chromophore. The absence of the 245-nm disulfide Cotton effect allows no direct Fig. 3. Temperature dependence of the Cotton effect of observation of the interconversion of the P-helical cystine diketopiperazine at 225 nm in O.lN HC1 diastereomer into the M-helical isomer and vice (o o), in trifluoroethanol (a a), and in versa. However, the amide transitions should also ethanol (· ·). depend largely on these temperature-induced trifluoroethanol, the bends observed in the teminterconversions. Indeed, a rise in temperature perature dependence differ by more than 10 °C from 0 to 55 °C reduces the molar ellipticities at (Fig.3). This fact, and the observation of larger 222 nm to about 38% and at 194 nm to 26%. ellipticities of 1 in trifluoroethanol, indicate that Fig. 3 shows part of the sigmoid curve obtained this more strongly hydrogen-bonding solvent by plotting the standardized ratios of the molar stabilizes one of the two diastereomers. The dioxellipticities [ ]t/[ ]55 versus the corresponding ane, ethanol and methanol solutions show temperature t. smaller molar ellipticities for both Cotton efBelow 40 °C the thermodynamically more stabile fects. As expected, the η - π* Cotton effect of P-helical diastereomer predominates, and with theamide groups shifts hypsochromically with rising temperature, the population of the conincreasing solvent polarity from 231 nm in dioxformation with an M-helical disulfide group, prob- ane to 222 nm in trifluoroethanol (Table 1). A ably exhibiting a less negative Cotton effect, inhypsochromic shift is also observed for the posicreases. Above 40 °C there is only a minor detive short-wavelength Cotton effect. A weak crease in the ellipticity ratio due to the balanced Cotton effect was discovered at 305 nm in mepopulation of the two conformations. thanol and dioxane solutions of 1 (Fig. 2). Its ellipticity and position have only minor temperSolvent dependence of the CD of cystine ature and solvent dependence. Since different diketopiperazine batches of highly purified crystals of 1 have been A linear decrease in ellipticity of the η — τι* used in several independent measurements, the Cotton effect is observed in 0.1 N HC1 between origin of this new Cotton effect is certainly not 20 and 100 °C (Fig.3). In ethanol (100%) and in due to an impurity. It is possible that a positive
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1016 Table 1. Position and molar ellipticities of the Cotton effects of cyclo-L-cystine.
M. Ottnad, P. Hartter and G. Jung
Solvent
Temp. [°C]
Dioxane
20
Methanol
20
Ethanol(95%)
25
0.1NHC1 Trifluoroethanol
20 20
Cotton effect above 300 nm arising from a chargetransfer transition is superposed by the negative amide Cotton effect to such an extent that a positive maximum below 300 nm appears. A similar phenomenon is observed in the case of 6,8-thioctic acid1101. The different perturbation of the amide transition in the Ρ and M-helical arrangements induces different CD band contours in the two forms. As a consequence, the CD band of one conformation is superposed by the flatter band of the other form, resulting in the weak positive Cotton effect at 305 nm. This explanation is supported by the symmetric shape of the 305 nm band. Referring to studies with epidithiadiketopiperaznW41', it seems reasonable that a CT transition transfers an electron from an occupied na, nb sulfur orbital into the antibonding orbital of the amide groups of 1. Such a CT transition may be possible because of the close proximity of the sulfur atoms and the carbonyl groups in the P-helical form. Because of the 250 nm disulfide absorption, such a transition is not observed. However, the absence of a solvent dependence contradicts this assumption, so further experiments will be necessary in this context. Time dependence of the CD ofcystine diketopiperazine Because of the relatively high rotational barrier between the conformational isomers la and Ib, it should be possible to detect one of these two forms by measuring freshly prepared solutions of 1, provided the crystal embeds only one form. In the first paper on the conformation and configuration of cyclo-L-cystinel7], it was pointed
Bd. 356 (1975)
λ (nm] 305 231 305 226 197
227,5 198,5 224 222 194
[ o j [degxcm 2 xdmol~ 1 ] 31 51480 16 56 000 327070 51100 259 850 - 52870 - 62130 360 340 -
out that the M-helical form could possibly exhibit a smaller negative (or even slightly positive) rotational strength .R than the P-helical diastereomer. Due to the influence of the -CH2-S- group in a positive octant relative to the peptide carbonyl chromophor, la should possess the higher and negatived when compared to Ib. The spectroscopic registration of an excess of either la or Ib directly after dissolution of the crystals before equilibrium is reached should give information on the screw sense in the crystalline state. However, such a conclusion can be equivocal when the Mhelical form Ib possesses a stronger overall rigidity, including solvent shell influences. Further complications may arise when 1 dissolves in an aggregated state, which generally exhibits stronger Cotton effects than the corresponding monomers. In this case, a decrease in ellipticity will be found as long as the oligomers dissociate into monomers. Table 2 shows the results of a first tentative experiment on the time dependence of the CD after dissolving 1 in 95 % ethanol at 4 °C and 25 °C. Indeed, a systematic change in the intensity with time is found. The decrease in ellipticity is expected for a change from a predominantly P-helical population to aP- and M-helical mixture, not considering possible restrictions. The decrease in ellipticity of the η — π* transition at 227 nm is finished after about three days at 4 °C, corresponding to a total decrease of 15 - 20%. In all previous and common experiments investigating the temperature dependence of the CD of 1, a normal decrease with increasing temperature was found. Because the distribution of the diastereomers comes to an equilibrium faster at 25 °C, the time dependence is less pronounced in
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Table 2. Time-dependence of the CD after dissolving crystals of cystine diketopiperazine.
Time [h]
[Θ] [degx cm2 χ dmol
Solution in 95%ethanol at4°C \ max = 227 nm
0.5 20 46 68
-
Solution in 95%ethanolat25°C
1 21
- 54000 - 53200
this case. However, it is understood that many of the CD data obtained by us and others can be superimposed by a time dependence. A detailed spectroscopic kinetic analysis of these time-dependent phenomena, including an investigation of solvent, temperature and possible concentration effects, will be reported elsewhere. As yet the experiments should by no means be considered as indicative of a partial Ρ -> Μ transition of 1 going from crystal to solution, but as a new and promising technique which has already been very useful in similar cases^31'. Temperature dependence of the 13CNMR of cystine diketopiperazine Several aspects of the exact estimation of energy barriers by pulsed NMR techniques are problematic and require a complete line shape analysis142"441. Since the population of the less stable M-helical conformation Ib is relatively small, a broadening of the line width could not be observed because of the small S/N ratio of the Ib signals (Fig.4, Tab.3). Table 3. Temperature dependence of the
l
]
56250 51950 48 900 47850
However, an improved S/N ratio requires very long accumulation times, which are intolerable, in particular at high temperature. Below 40 °C there are three weak carbon signals besides the three intense signals of the predominating diastereomer, giving proof for the exact C2 symmetry of both forms*71. The stronger signal of the carbonyl carbon is found at lower field. This indicates, independently from the CD, that there is a close proximity between the sulfur atoms and the carbonyl group in the P-helical diastereomer la. The deshielding of the carbonyl group in la could be caused by a through-space electric field effect of the 3p lone pair electrons of the sulfur atoms shifting the signal to lower field than the corresponding signal of the M-helical diastereomer Ib. Large through-space effects of the sulfur of cysteine to its C = 0 group are long known to us from the pH and temperature dependence of the 13C NMR of oxidized and reduced glutathionef30'45'461 and of cysteine and related aminothiols130'471. Solvent shell influences must be taken into account for the ob-
13
C NMR signals of cystine diketopiperazine in [2H6]Me2SO. 13
Temp. [°C] Cys-CO 25 28 35 40 45 50 55 65 75 98
1017
Sterically Hindered Bisulfide Bridges
Bd. 356(1975)
Ρ
Μ
170.69 170.67 170.67 170.47 170.47 170.47 170.36 170.25 170.14 169.39
166.81 166.67 166.67
C chemical shift δ [ppm]Me4Si = 0 Cys-Ca Μ Ρ 56.32 56.31 56.31 56.31 56.31
53.84 53.84 53.73 53.73 53.84 53.73 53.73 53.73 53.84 54.05
Cys-C0 Ρ
47.58 47.58 47.58 47.47 47.47 47.37 47.37 47.26 47.14 46.71
Μ
44.67 44.67 44.67 44.67 45.64
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1018
M. Ottnad, P. Hartter and G. Jung
Bd. 356(1975)
z
u
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Bd. 356(1975)
Sterically Hindered Bisulfide Bridges
served shift differences of the two diastereomers, although these are considered in general to be small. However, we observed common upfleld shifts of 0.6 to 1.5 ppm of C and C = 0 resonances of amino acids in the lipophflic octadecapeptide antibiotic alamethicin in going from chloroform or methanol solution to the strongly polar dimethylsulfoxide'481. Therefore, the observed upfield shifts of the Cys-COand Cys-C^ in cystine diketopiperazine in changing from the P to the M-helical disulfide arrangement may be also due to a small extent to solvent influences. An increased solvent-solute interaction is expected for the M-helical diastereomer Ib, which has more exposed peptide carbonyls. As expected, the conformational inner molecular changes and accompanying solvent influences shift the position of the Cys-Ca to a minor extent. Whereas Cys-C^ and Cys-CO shift to higher field, in the case of C a , a reverse shift to lower field is found for the M-helical form. The somewhat larger shift difference for the peptide carbonyls (Δ = 3.9ppm) with respect to Cys-C^ (Δ = 2.5 ppm) and Cys-C^ (Δ = 2.9 ppm) is not unexpected (Tab.3). It has long been known that sp2 hybridized carbons in general show the largest shift effects on electrostatic changes of their environment'491. The theoretical relationships between electron density and carbon chemical shifts are not very satisfying as yet'49'501. Particularly in the case of the complex dissociation equilibrium of thiols and disulfides, a clear-cut differentiation between the through-space electric field effects expected to produce downfield shifts'511 and the inductive through-bond effects1521 acting in the opposite direction is rather difficult^47'531. However, our experiments so far indicate particularly large through-space influences of the sulfur 3p orbitals on nearby carbons, which often give rise to 13C chemical shifts opposite to those observed in corresponding non-sulfur analogs. In the present case, the optical measurements prove an interaction between the disulfide and carbonyl chromophores which changes the excitation energy of the η-π* transition. It is evident that this perturbation also has a strong influence on the paramagnetic shielding σρ of the carbonyl 13C nuclei. In the case of the diketopiperazine of cystine, a rise in temperature results only in a relatively small shift (Table 3) when compared to the tem-
1019
perature-induced shifts observed for the cystine carbons in oxidized glutathione'301. Above 50 °C, the smaller signals of the M-helical diastereomer disappear and the interconversion of la and Ib occurs so rapidly that one observes only single signals. These signals shift with further increase of the temperature due to the decrease in viscosity and solvent/solute interactions. This investigation is the first determination of an energy barrier in a peptide and a disuifide using 13 C NMR. Although we are aware of all the advantages of this method and all the objections concerning the reliability of the determination of energy barriers from pulse Fourier-transform 13C NMR spectra, we estimated the activation enthalpy of the interconversion. From a logarithmic plot'421 of Δι;, the difference of the 13C resonances of the two conformers (expressed in Hz), versus Δ G # with T(° K) as parameter, Δ0# values of 15.6 to 16.0 kcal/mol are obtained. In this context, it is interesting that the highest known value of another example of a an energy barrier in a hindered diaryl disulfide was found to be 15.7 kcal/mole'541. As for 1, this can be considered only as the lower limit for these processes. Unfortunately, our measurements are restricted as yet to Me2SO solutions because of the low solubility of 1. From the intensities of the 13C NMR signals of la and Ib, the percentage of the less stable M-helical form Ib can be estimated to be between 15 and 20% at 25 °C. This estimation is reliable, since the relaxation times and the nuclear Overhauser enhancements of the aliphatic carbons in both forms should be comparable. Measurements of the longitudinal relaxation times Τ ι well above the coalescence point, about 60 °C, and below, about 0 °C, should reveal further evidence of the intramolecular motion of 1. Circular dichroism of cysteinyl-cysteine disulfide and derivatives The hydrolysis of one of the two cis amide bands in the homodetic and heterodetic, bicyclic cystine diketopiperazine 1 yields the heterodetic monocyclic cysteinyl-cy steine disulfide 2. Conformational calculations for 2 predict a non-planar cis amide bond and bond lengths and angles similar to those of open chain cystine peptides. The con-
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1020
M. Ottnad, P. Hartter and G. Jung
formational energies of the P and M-helical forms of 2 should differ only by about 0.8 kcal/mole[361. Therefore a CD spectrum with a relatively weak Cotton effect around 250 to 265 run, exhibiting a temperature dependence similar to that of glutathione^22' or cystine, is expected. In contrast to this expectation, we observe a spectrum similar to that of 1, except for the opposite signs and lower eilipticities of the Cotton effects (Fig. 5). The differences are due to the fact that the single amide bond in 2 has no dipole-dipole interactions with a second one, as is the case for 1. The reduced eilipticities are partly due to the higher flexibility of 2. The solvent dependence of the CD of 2 shows that the positive Cotton effect at 225 to 230 run originates from an amide transition, and the short wavelength CD band has the same origin as in 1 (Tab.4). The long wavelength,
Bd. 356 (1975)
negative and weak Cotton effect is due to a disulfide transition, because it shows a large temperature dependence in methanol and in 0.1 N HC1, and also a bathochromic shift with increasing temperature. The temperature-induced decrease in the eilipticities at 200 and 227 nm is relatively small. The weak negative maximum of the longest wavelength Cotton effect may be due to an overlapping of a disulfide transition with the strong positive amide CD band. Therefore a position at much longer wavelength is found when compared to glutathione or cystine, and also the bathochromic shift of this maximum with increasing temperature is readily explained. The CD spectra of 3 are almost identical with those of 2 (Fig. 5) in the short-wavelength region. However, due to the presence of the ester group, there are differences in the long wavelength region. As with 1, there are no long-wavelength
Fig. 5. Circular dichroism spectra of f-butyloxycarbonyl-L-cysteinyl-cysteine-disulfide methyl ester (4) ( ), L-cysteinyl-cysteinsdisulfide methyl ester hydrochloride (3) ( ) and L-cysteinyl-cysteine-disulfide hydrochloride (2) ( ) at 20 °C in absolute ethanol.
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Bd. 356 (1975)
Sterically Hindered Bisulfide Bridges
Cotton effects in strongly polar solvents, e.g., O.lN HC1, trifluoroethanol and hexafluoroacetone trihydrate. The negative Cotton effect at 285 nm found in methanol shows the same temperature dependence as the corresponding band of 2, an apparent CD maximum of a disulfide transition overlapping with the strong and positive amide band. This is confirmed by the shoulder found in the ultraviolet spectrum of 3 at 245 nm corresponding to the absorption range of disulfides with torsional angles around 90°. The CD spectrum of 3 in absolute ethanol shows a small positive Cotton effect at 305 nm that has also been found for cystine diethyl ester in propanol solution. It is not possible to predict the helicity of the disulfide groups of 2 and 3 from the CD, since the ellipticities of the Cotton effects are too small, and they may be largely influenced by effects of their environment. The introduction of the bulky f-butyloxycarbonyl group gives the fully protected dipeptide 4, which exhibits a CD spectrum in absolute ethanol (Fig. 5) deviating considerably from those of 2 and 3. The two long wavelength Cotton effects at 260 nm (+) and 243 nm (—) are due to a disulfide transition. They are situated symmetrically to 257 nm. This may be due to a torsional angle similar to that of open-chain disulfides. These assignments of the long-wavelength CD bands are supported by the finding that N,N'-dimethyl-L-cystine also exhibits two CD bands at 280 nm (+) and 247 nm (-) in 0.1 N HC1. Therefore the successive introduction of substituents on the amino group seems to induce a common effect on the CD of cystine derivatives. This band splitting is also observed in chiraldialkyldisulfidesl5]. The change from methanol or ethanol to the less polar solvent dioxane reduces the ellipticities of the long-wavelength bands drastically and gives a bathochromic shift of 40 nm. On the other hand, the ellipticity of the amide band is severalfold increased. This effect is also observed on the heterodetic monocyclic tripeptide disulfides Boc-Cys-X-Cys-OMe [X =[38 Gly, Ala, Leu, Phe, Val,Glu(OBuO,Lys(Z)] K * Ottnad, M., Hartter.P. & Jung, G., unpublished results.
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