1 NMR characterization of peptide bonds in linear and cyclic peptides Darrien James and Markus Germann Georgia State

University, Atlanta, GA 30303 Abstract NMR spectroscopy is used to study the peptide bond conformations in the linear and cyclic forms of the tetrapeptide IGGN. Five minor peptide bond conformers were discovered in the linear peptide. However, only a G3 peptide bond minor conformer was observed for the cyclic tetrapeptide. At 293K, 0.41 % cis G-3 peptide bond isomer population was detected for the linear tetrapeptide. The trans to cis isomerization of the G-3 peptide bond is exothermic. The enthalpy of the process is 35.5 kJ/mol. It was also found that the rate of the isomerization increases with increasing temperature.

Introduction Peptide bonds form the backbone of protein chemistry. The amino acid sequence of a protein contains important folding information. These unique folding patterns define the function of the protein.1 A peptide bond has partial double bond character rendering the bond planar. The electrons of the carbonyl bond are delocalized via resonance (figure 1), consequently, the bond is rigid but some rotation is possible.2 This characteristic allows us to study the cis (Z) /trans (E) isomerization at peptide bonds.3 NMR spectroscopy can be employed in the study of the conformational change of peptide bonds. 4

Figure 1. Illustration of the peptide bond resonance.

Currently, most academic and pharmaceutical labs study peptides in their trans form. Our research in cis peptide bond analysis could provide the foundation for a more holistic study of peptides. Moreover, most small peptide drugs have trans peptide bonds. Therefore, investigating the cis isomer will further diversify peptide drug chemistry. Proteases naturally metabolize trans peptide bonds. Therefore, peptide drugs with their peptide bonds locked in the cis conformation could be less susceptible to enzymatic degradation. This innovation could potentially lead to a substantial improvement in the bioavailability of peptide drugs.

Prolyl peptide bonds have been studied extensively. Proline residues are common at the edge of beta strands and as the 1st residue in alpha helices. In a typical peptide bond the trans isomer is preferred greatly over the cis isomer. However, the trans peptide bond is only slightly preferred in a prolyl peptide bonds. This is due to the fact that the nitrogen of proline is bonded to two tetrahedral carbon atoms.5

The proline cis isomer population of an unstructured peptide is 5%-50%. 6 The amount is dependent on the amino acid that is adjacent to the proline. Prolines flanked by aromatic

3 residues have higher cis content when compared to prolines flanked by residues of other amino acids. This is due the steric restrictions generated by the large aromatic side chains of tyrosine, tryptophan, and phenylalanine. Reimer found that the unstructured oligopeptide acetyl-Ala-Xaa-Pro-Ala-Lys-NH2 showed proline cis populations were highest for Trp-Pro peptide bonds, 38% and lowest for Pro-Pro, 6%. 7

While, researchers studying non-prolyl peptide bonds report a cis secondary amide peptide bond population ranging from 0.1% -1.00% for the following peptides: Ala-Tyr, Tyr-Ala, Phe-Ala, Ala-Phe, Ala-Ala-Tyr, Ala-Ala-Tyr-Ala and Ala-Ala-Tyr-Ala-Ala. The cis-trans isomerization was quantified by NMR line shape analysis and magnetization- transfer experiments. The 1H NMR analysis of Ala-Tyr was carried out at different temperatures. The chemical shifts for the cis and trans methyl protons of alanine in Ala-Tyr were recorded at 369 and 295K. The first run was performed by gradually raising the temperature to 369K. The line broadening and signal shifts increased gradually. At 295K, the line shapes and signal ratios of the alanine methyl protons were identical to those observed at 369K. New signals were observed as time progressed at higher temperatures. However, unlike the cis and trans methyl protons of alanine, these new minor conformers, due to partial decomposition products, showed no reversibility. Consequently, Scherer et al. were able to infer that the conformational isomerism of the dipeptide Ala-Tyr rendered the alanine methyl group chemically different in both conformers. Exchange spectroscopy provided further evidence of two inter-converting Ala-Tyr species. Thereafter, Scherer uses this evidence and data as a model to study the secondary peptide bond isomerization of the

4 Ala-Xaa moiety in all the peptides mentioned above. It was found that the cis populations were dependent on chain length and ionization state of the peptides. 8Similarly, our research involves the study of peptide bond conformations. However, our efforts are focused on the cis (Z) / trans (E) isomerization at the primary peptide and not the secondary peptide bond. 1H and 13C NMR spectroscopy are used to determine the population of the cis isomer present in a solution of the linear tetrapeptide with sequence IGGN. NMR is also used to explore if conditions can be found that increase the population of the cis isomer. NMR along with molecular modeling software is used to determine the structure of the cyclic peptide IGGN. It is believed that all the peptide bonds in the cyclic peptide are in the trans form. This is due to the conformational restriction produced by the ring.

STRUCTURES OF THE LINEAR AND CYCLIC TETRAPEPTIDES I1-G2-G3-N4

6 Procedure
Instrumentation All NMR experiments were performed on a Bruker Avance 500 MHz spectrometer operating at a 500.18 MHz proton frequency. A 5 mm TBI probe head was used throughout the study.

Assignment The assignments for both peptides were determined with 1-dimensional and 2dimensional NMR experiments. These techniques include 1D proton NMR, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence spectroscopy (HSQC), nuclear overhauser effect spectroscopy (NOESY) and heteronuclear multiple bond correlation spectroscopy (HMBC).

COSY experiments provided connectivity information for neighboring protons separated by 3 bonds. The TOCSY experiment provided further connectivity information going down the amino acid chain. In all TOCSY experiments the mixing time was set to 80 ms.

The NOESY technique was used to assign resonances that could not be assigned by other experiments. The experiment requires that the atoms are close in space. The atoms must be within 5-6 .9 The technique was also used to support other chemical shift assignments obtained using correlation experiments. The NOESY mixing times that were used in the cyclic and linear chemical shift assignments were 50, 75, 150 and 300 ms. A 50 ms experiment was performed on the linear peptide while a 75 ms experiment was performed on the cyclic peptide. The following heteronuclear experiments were performed to obtain data on C-H connectivity, long-range carbon to hydrogen coherence and nitrogen chemical shifts. The

7
13

C HSQC provided data on C-H connectivity. While, the 15N HSQC provided the

nitrogen chemical shifts for the cyclic peptide. The HSQC, was indirectly referenced using the method outlined by Wishart et. al.10 The HMBC technique facilitates the determination of long range (two and three bond) H- heteronucleus connectivity. It was employed in the determination of long-range carbon to hydrogen coherence in the cyclic peptide. The experiment was used to assign the carbonyl chemical shifts for the cyclic peptide.11 Linear peptide A 6.5 mM solution of the linear peptide was prepared by dissolving 2.6 mg of the peptide in 1 mL of DMSO. Initially, 1D and NOE spectra were used to detect minor conformers of all four peptide bonds. In the NOE spectra, each peptide bond had 1 or more exchange peaks with smaller diagonal peaks within the amide region (figure 3). These peaks were identified as the minor conformers of the peptide bonds. The signal intensities of the peaks were measured and tabulated. However, the G-3 peptide bond was chosen for further 1D peptide bond characterization studies because it had the strongest NOE exchange peaks at a temperature of 298K and a mixing time of 300 ms. Four 1H NMR experiments were performed at the following temperatures 293, 303, 308, and 313K. The peak areas for the major and minor conformers of the G-3 peptide bond were then calculated using the integration function. The signal of the major conformer was used as the calibration peak. It was assigned an area of 1 then the minor conformer peak was integrated. These areas were used to determine the equilibrium constant for the isomerization at each temperature. Thereafter, the free energy, enthalpy and entropy changes involved in the isomerization were calculated from the equilibrium constants.

Cyclic Peptide A 6.3 mM solution of the cyclic peptide was prepared by dissolving 1.8 mg of the peptide in 0.6 mL of DMSO. Thereafter, a more concentrated 30mM solution was made to obtain better signal to noise in NMR experiments. The Sparky program was used to assign and integrate NOE cross peaks.12 Cross peak intensities obtained from the distance map were then used to calculate the distances between all hydrogens that produced NOE signals. Several models of the cyclic tetrapeptide were constructed using PCModel. The first had all peptide bonds in the cis conformation, while the other two models each had 1 of the glycine peptide bonds in the cis conformation. There after a series of models were constructed in which the carbonyl orientations were varied.

Results The chemical shift assignments for the linear and cyclic peptides were mostly routine. However, differentiating the 2 glycines was more difficult. NOE experiments allowed for the differentiation between the glycines. It was also interesting that the alpha hydrogens of the glycines were degenerate in the linear but not in the cyclic peptide. This provided evidence of the cyclic peptides conformational restriction. Also, for resonances with 2 protons such as alpha hydrogens of the glycines, the hydrogen with the lower chemical shift was named alpha-1. Similarly, for the beta and gamma hydrogens of isoleucine, the hydrogens with the lower chemical shift were named beta-1 and gamma-1. The same convention was used for naming the beta hydrogens of asparagine. The chemical shift assignments are in the tables below.

9 THE LINEAR TETRAPEPTIDE I1-G2-G3-N4

Ile (I1) N-H H1 H2 H1 H2 H1 H2 H' H NH2-1 NH2-2 Term. NH2-1 Term. NH2-2 Acetyl H 7.92 4.12 1.40 1.08 1.08 0.81 0.80
1 13

57.0 24.3 24.3 15.0 11.0

Gly (G2) 13 H C 8.25 3.71 42.0 3.71

1

Gly (G3) 13 H C 8.08 3.68 42.0 3.68

1

Asp (N4) 13 H C 8.03 4.46 50.0

1

2.50 2.39

37.0

6.85 7.27 7.04 7.10 1.87 22.2

Table 1. NMR chemical shifts of the linear peptide hydrogens and carbons in DMSO at 298K.

THE CYCLIC TETRAPEPTIDE I1-G2-G3-N4

Ile (I1)
1

Gly (G2)
15

Gly (G3)
15

Asp (N4)
15

13

13

13

13

15

N-H H1 H2 H1 H2 H1 H2 H' H NH2-1 NH2-2 C=O Amide N N

7.68 3.92 1.81 1.14 1.47 0.81 0.78

58.1 36.0 25.0 15.0 10.0

7.75 3.51 3.92

42.0

8.67 3.58 3.72

43.0

8.32 4.46 2.44 2.39

50.0 36.0

6.93 7.33 171.7 110.0 170.9 99.6 169.0 104.2 171.1 113.7 104.3

Table 2. NMR chemical shifts of the cyclic peptide hydrogens and carbons in DMSO at 298K.

10

Figure 2. 1H NMR spectra of the linear tetrapeptide within the amide proton region at 298K in DMSO. The intensity of the G-3 minor peak was far lower than that of the major N-H signals as a result the two spectra with different peak height scales were superimposed.

G - 3 minor

G3 G2

N4

I1

Figure 3. NOESY spectra of the amide protons of the linear tetrapeptide at 308 K with a mixing time of 150 ms in DMSO.

11

Minor conformers were detected for all four peptide bonds. Interestingly, two exchange peaks were observed for the G3 peptide bond. However, only 1 was identified as being due to a minor conformer. This G3 minor conformer is labeled in the spectrum above.

I-1 G-2
exchange peak

N-4
exchange peak

G-3

Figure 4. NOESY spectra of the amide protons of the cyclic tetrapeptide at 308 K with a mixing time of 150 ms in DMSO.

In the exchange spectrum above the minor conformer of the G3 N-H proton of the cyclic tetrapeptide is shown. Though the diagonal peak of the minor conformer was not completely resolved, the cross peaks in both dimensions provide evidence that there is a minor conformer of the G3 N-H proton with a chemical shift of 7.95 ppm. It is hypothesized that this minor conformer is the cis G-3 peptide bond. Since, the tetrapeptide has very little conformational mobility.

12

STRUCTURE OF N-METHYLACETAMIDE

TRANS

CIS

EMM = -7.43 kcal/mol

EMM = -0.035 kcal/mol

QM Chemical Shift Calculations (ppm) Trans 4.89 Cis 4.66

Table 3. Spartan QM Chemical shift calculations for the cis and trans conformations of a peptide bond amide hydrogen in n-methylacetamide using the B3LYP6-31G** density function.

The quantum mechanical calculations above supported the 1D chemical shift order of the G-3 N-H major and minor conformers (figure 2). Both procedures showed that the chemical shift of the major conformer was higher. The minimization energies were calculated in Hyperchem. The MM+ force field and the steepest decent minimization algorithm were employed.

13

Figure 5. Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The above curve illustrates that the amount of the cis isomer decreases with increasing temperature. The R2 value for the curve is 0.9391.

T (K) 293 298 303 308

a b

% Cis 0.41 0.39 0.27 0.21

Fraction Cis 0.0041 0.0039 0.0027 0.0021

Fraction Trans 0.99996 0.99996 0.99997 0.99998

K 4.1 x10-3 3.9 x10-3 2.7 x10-3 2.1 x10-3

ln (K) -5.50 -5.55 -5.91 -6.17

1/T (1/K) 3.41 x10-3 3.36 x10-3 3.30 x10-3 3.25 x10-3

G a (J/mol) 13399 13751 14889 15801

H (J/mol) -35528 -35528 -35528 -35528

S b J/mol-K -205.2 -203.6 -204.7 -204.9

From the free energy equation G = -RTlnK From the free energy equation G = H-TS

Table 4. Thermodynamic data for the G-3 peptide bond isomerization in DMSO.

The calculated Gibbs free energies were all positive therefore the isomerization is nonspontaneous between 293 and 308K. It was found that the percentage cis G-3 NH increased as the temperature was lowered. Therefore, the G3 trans cis isomerization for the linear tetrapeptide IGGN is an exothermic process. The calculated enthalpy for the G3 trans cis isomerization was 35.5 kJ/mol.

14

Figure 6. G-3 peptide bond Isomerization: Van Hoff Plot: ln (K) as a function of reciprocal temperature.

The following equation, lnK = -H/RT + S/R, was used to construct a Van Hoff plot for the isomerization. The lnK was the y term while 1/T was the x term. The slope = H/R= 4273.6 K. This value was then was then multiplied by the gas constant to give the enthalpy of the isomerization, H= -35.5 kJ/mol. Van Hoff Curve Equation: y = 4286.4x - 20.047 R2 = 0.9297.

15
Temperature Mixing Time Amide (K) (ms) Proton G-2 Major Peak 8.8x10
6

Exchange Exchange Peak A Peak B 7500

293

150

G-3

9.2x10

8400

7700

N-4

7.8x10

6400

G-2

10.0x10

9592

298

50

G-3

11.0x10

8800

7012

N-4

9.0x10

6212

G-2

8.6x10

12000

298

150

G-3

8.6x10

12000

10000

N-4

7.6x10

11400

G-2

12.5x10

32000

298

300

G-3

12.6x10

33000

26000

N-4

12.0x10

24000

G-2

7.5x10

20540

308

150

G-3

8.2x10

21000

21000

N-4

7.2x10

18000

Table 5. Intensity of NOESY spectra exchange peaks for the major and minor conformations of the peptide amide bond protons. All of the above NOESY experiments were done in DMSO.

16

This NOESY analysis was done to get the rate of the trans-cis isomerization. The peak intensities were measured using both the NOESY and 1D experiments. The exchange peaks labeled A are for the trans-cis isomerization of I1, G2, G3 and N4 amide hydrogens with the corresponding diagonal peaks. The exchange peak labeled B was only detected for G-3 peptide bond. Currently, we are not sure of the identity of the diagonal peak that corresponds to the exchange peak labeled B. It could be 1 minor form inducing another minor form. Isomerization Rates
mt (s) 0.01 0.02 0.03 0.05 Xa 0.998 0.998 0.998 0.998 Xb 0.0021 0.0021 0.0021 0.0021 Iaa Ibb 7 5 6.70x10 1.41 x10 7 5 6.00 x10 1.26 x10 7 5 6.10 x10 1.28 x10 6.00 x10
7

Iab 33000 39000 73000 70000

Iba 14000 38000 41000 70000

r 10.9 5.54 3.49 2.60

k ct (s-1) 120.1 72.02 60.06 44.98

k tc (s-1) 0.252 0.151 0.126 0.095

1.26 x10

Table 6. G3 peptide bond isomerization data.

A model developed by Perrin et .al was employed to calculate the rates of isomerization of the G3 peptide bond at 308K. The formula below shows how the data in the table above was computed.13

Eqn. 1

Eqn. 2

17

Perrins method proved to be ineffective for this system because the relative population difference was too vast. However, other appropriate models will be tested and implemented.

Rates analysis Seven NOE experiments with different mixing times were carried out at 308 K to examine the intensity change of the cross peaks between the major N-H diagonal peaks and the smaller cis conformer peaks (figure 3 labeled NOE) The mixing times were 50 ms, 100 ms, 250 ms, 500 ms, 750 ms, 1000 ms and 1500 ms. The diagonal peak intensities all decreased as the mixing time was increased. While, the cross peak intensities increased to a maximum at a specific mixing time, thereafter, their intensities decreased. The following curves summarize the diagonal and cross peak development as a function of mixing time.

18

Figure 7. NOE Intensity of the I1 N-H diagonal peak as a function of mixing time.

Figure 8. NOE Intensity of the G2 N-H diagonal peak as a function of mixing time.

19

Figure 9. NOE Intensity of the G-3 N-H diagonal peak as a function of mixing time.

Figure 10. NOE Intensity of the N-4 N-H diagonal peak as a function of mixing time.

20

Figure 11. NOE intensity of I1 N-H exchange peak A as a function of mixing time.

Figure 12. NOE intensity of I1 N-H exchange peak B as a function of mixing time.

21

Figure 13. NOE intensity of G2 N-H exchange peak A as a function of mixing time.

Figure 14. NOE intensity of G2 N-H exchange peak B as a function of mixing time.

22

Figure 15. NOE intensity of G-3 N-H exchange peak 1A as a function of mixing time.

Figure 16. NOE intensity of G-3 N-H exchange peak 1B as a function of mixing time.

23

Figure 17. NOE intensity of G-3 N-H exchange peak 2A as a function of mixing time.

Figure 18. NOE intensity of G-3 N-H exchange peak 2B as a function of mixing time.

24

Figure 19. NOE Intensity of the N4 N-H exchange peak A as a function of mixing time.

Figure 20. NOE Intensity of the N4 N-H exchange peak B as a function of mixing time.

25

Figure 21. T1 Inversion Recovery plot of the G-3 N-H minor form diagonal peak as a function of delay ().

The 1D 1H NMR intensity of the minor form of the G-3 NH resonance obtained from a T1 IR experiment was plotted as a function of delay to determine the spin-spin lattice relaxation time (T1). The T1 value was obtained from a curve fitting to the general equation y= m1*(1-2*e-x/m2). The T1 value for the minor form of G3 N-H was 583 ms. This value is close to the average T1 obtained for the G3 N-H major conformer, 623 ms. The table below summarizes the T1 values obtained for the N-H protons at 293, 298, 303, and 308K.

26

Temperature (K) 293 298 303 308

N-H I1 G2 G3 N4 I1 G2 G3 N4 I1 G2 G3 N4 I1 G2 G3 N4

T1 (ms) 600 538 627 600 597 528 615 582 607 534 618 584 624 537 632 597

Table 7. Spin-spin lattice relaxation times for the N-H protons of the linear tetrapeptide at various temperatures.

Structural Elucidation of the Cyclic tetrapeptide IGGN

NOESY spectra were used to determine the distance between the hydrogen atoms in the cyclic tetrapeptide I1-G2-G3-N4. The distance between NH2-1 - NH2-2 amide hydrogens of the asparagine was used as the reference distance. The table below shows the NH2-1 NH2-2 distance measured from a model of the cyclic peptide constructed in PC Model, alongwith, the NOE cross peak intensities for NH2-1 - NH2-2 at three mixing times.

27 The NH2 -1- NH2-2 amide reference distance, the NOE cross peaks intensities for NH2 -1- NH2-2 and the individual resonances were substituted into the equation below to calculate all the tabulated distances. All reported distances are in angstroms ().

Ia / Ib

rb 6 / ra 6

Eqn. 3

Mixing Time (ms) 75 150 300

NOE Intensity Distance () 6 5.64 x 10 1.65 6 7.47 x 10 1.65 6 6.38 x 10 1.65

Table 8. NOE reference data collected at three different mixing times.

NH-NH

N-H N-H
75 150 300

I1 I1 75 150 300 G2 1.9 2.0 2.1 G2 75 150 300 G3 ne 5.2 4.5 3.2 3.1 3.0 G3 75 150 300 N4 2.6 2.6 2.5 3.9 3.7 3.7 3.1 3.1 3.2 N4

Table 9. NH-NH distances calculated at 3 different mixing times. The abbreviation ne means there was no peak.

28

H- NH N-H H
75 150 300 75 150 300 75 150 300 75 150 300

I1 3.92 I1 2.7 2.5 2.7 1-n 2-n 1-n 2-n 1-n 2-n 1-ne 2- n 1-ne 2-3.5 1-ne 2-3.5 2.7 2.6 2.6 G2 ne + ne + ne 1- n 2-2.4+ 1-2.4 2-2.3+ 1-2.3 2-2.2+ 1-n 2-n 1-n 2-n 1-n 2-n 3.8 4.4 4.2
+

H s

G2 3.51, 3.92 G3 3.59,3.72 N4 4.46

G3 ne ne ne 1-n, vc *2-2.3 1-n *2-2.3 1-n *2-2.2 1- n, vc 2- 2.6 1-2.2 2- 2.5 1-2.2 2- 2.7 ne ne ne

N4 ne ne 3.9 1-ne 2-ne 1-ne 2-ne 1-ne 2-ne 1-3.0 2-2.3 1-2.8 2-2.3 1-3.1 2-2.2 c 2.4 2.4

Table 10. H-NH distances calculated at 3 different mixing times. The following abbreviations are used
in the above tabulation: ne- none: there were no cross peaks, n-low S/N, c-coupling: spin coupling peak distortion, and vc- very close to the diagonal: the NOE cross peak is too close to the diagonal to obtain accurate volumes. *+ I1H and G2H2 are isochronous; both hydrogens have the same chemical shift, as a result, molecular modeling applications were employed to differentiate the alpha hydrogens. The volumes obtained for these resonances with identical chemical shifts were compared with model distances to decipher the cross peaks.

29

H- All other Resonances All other Resonances

75 150 300 75 150 300 75 150 300 75 150 300 I1 -2.8 '-2.7 1- 3.2 2-3.7 I1 -2.8 '-2.5 1- 3.1 2-3.7 - 2.8 '-2.5 1- 3.0 2-3.6 ne G2 ne ne ne G3 ne ne ne N4 ne ne

G2 ne ne ne H- 1.8 H- 1.8 H- 1.8 ne ne ne ne ne ne G3 ne ne ne ne ne ne H- 1.6 H- 1.7 H- 1.7 ne ne ne N4 ne ne ne ne ne ne ne ne ne ne ne ne

Table 11. H -All other resonance distances calculated at 3 different mixing times. * I1H and G2H2 are isochronous. However, model evidence supports these distances are G2H1- G2H2. G31, G32 and G21 all have cross peaks with I1' however these very weak peaks were in the noise.

H All other Resonances All other Resonances H

I1 75 150 300 75 150 300 75 150 300 G2 '- 2.5 '-2.4 '-2.4 ne ne ne ne ne ne G3 ne ne ne ne ne ne ne ne ne H- 2.7 H- 2.8 H- 2.5 H- 2.4 H- 2.2 H- 2.6 N4 ne ne ne NH2-1- 4.2 NH2-1- 4.3 NH2-1- 3.6 NH2-1- n NH2-1- 4.2 NH2-1- 4.2 NH2-2- 2.9 NH2-2- 2.9 NH2-2- 2.8 NH2-2- n NH2-2- 4.2 NH2-2- 4.2

I1

H- 2.8 1-ps 2- 2.5 H- 2.8 1-2.9 2- 2.6 H- 2.8 1- 2.9 2- 2.4

N41

N42

Table 12. All H distances calculated at 3 different mixing times. The I1 and I1' resonances had similar chemical shifts; they are 0.78 and 0.81 respectively. The N41 - N42 cross peaks were very close to the diagonal and the peaks volumes were inconsistent.

30

NH All other Resonances

All other Resonances

G2 G3 N4
1- 3.8 1- 3.6 1- 3.4 2- 3.8 2- 3.6 2- 3.3

N-H

N-H
75 150 300 75 150 300 75 150 300 75 150 300

I1
-2.5 - 2.5 -2.4 - 3.0 - 2.9 - 2.7 1- 3.1 1-3.1 1- 2.9 2-3.3 y'-3.4 y2-3.2 y'-3.3 y2-3.1 y'-3.1 '- 3.3 '- 3.3 '- 4.1

I1 N-H

G2 N-H

G3 N-H
1- 2.6 1- 2.7 1- 2.6 2- 3.1 2- 3.1 2- 3.0

N4 N-H

Table 13. N-H- all other resonances distances calculated at 3 different mixing times.

The tables below summarize the N-H to N-H distance data obtained by varying the backbone carbonyl orientation of each amino acid in various models of the cyclic peptide. The models that were closest to the experimental distances obtained from the NOE experiment were G2 carbonyl down others up (table 18) and the G3 carbonyl down others up (table 17). This data suggests that the actual structure of the peptide could be similar to 1 of these models.
N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4 Model () 2.2 2.5 2.6 3.6 2.8 3.6 NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7 Absolute Difference 0.2 0.6 0.5 1.6 0.2 0.1

Table 14. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with all carbonyls pointing downward.

31

N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 4.1 4.1 4.1 4.3 4.1 4.3

NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 2.1 1.0 1.0 0.9 1.5 0.6

Table 15. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with alternating carbonyls.

N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 3.5 2.7 2.6 4.6 4.0 3.8

NOE (mt =150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 1.5 0.4 0.5 0.6 1.4 0.1

Table 16. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with N4 carbonyl pointing down and all other carbonyls pointing up.

N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 2.4 2.4 4.1 4.4 4.0 3.7

NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 0.4 0.7 1.0 0.8 1.4 0.0

Table 17. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G3 carbonyl down and all other carbonyls pointing up.

32
N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4 Model () 2.1 4.0 3.9 4.3 2.8 4.0 NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7 Absolute Difference 0.1 0.9 0.8 0.9 0.2 0.3

Table 18. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G2 carbonyl down and all other carbonyls pointing up.

N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 4.2 3.8 3.0 3.7 2.8 5.5

NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 2.2 0.7 0.1 1.5 0.2 1.8

Table 19. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with I1 carbonyl down and all other carbonyls pointing up.

N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 3.6 3.2 3.9 5.5 3.0 4.1

NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 1.6 0.1 0.8 0.3 0.4 0.4

Table 20. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G3 and N4 carbonyls down and all other carbonyls pointing up.

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N-H to N-H I1-G2 G2-G3 G3-N4 I1-G3 I1-N4 G2-N4

Model () 2.4 3.1 2.9 4.6 4.2 4.2

NOE (mt=150ms) () 2.0 3.1 3.1 5.2 2.6 3.7

Absolute Difference 0.4 0.0 0.2 0.6 1.6 0.5

Table 21. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with N4 and I1 carbonyls down and all other carbonyls pointing up.

Figure 22. A model of the cyclic tetrapeptide IGGN with G2 carbonyl down and other carbonyls up. This model has the best agreement with experimental N-H to N-H distances.

34 Discussion In the linear peptide, the concentration of the cis peptide bond isomers increased with increasing temperature. This result is consistent with earlier work conducted, which showed that the concentration of secondary cis peptide bonds increased with increasing temperatures for small peptides with the Ala-Xaa moiety. Remarkably, thermodynamic calculations revealed that the enthalpy of the G-3 peptide bond trans to cis isomerization is exothermic. It was hypothesized that the G3 peptide bond trans-cis isomerization would be an endothermic process since the cis conformer produces steric clashes between the oxygen and the hydrogen of the amide group. Furthermore, from the free energy equation, G = H-TS, the negative enthalpy and the positive free energy shows that the reaction is entropy driven.

While, the NOE rates data plots produced the expected trend; an exponential decay in NH diagonal peak intensity as a function of mixing time and an increase in the cross peak intensities until a maximum intensity was reached, thereafter, the cross peak intensities decreased. The T1 inversion recovery showed that the spin-spin lattice relaxation of all four N-H protons were similar. They ranged from 527- 632 ms.

The distance data obtained from the NOE experiments were reliable and made spatial sense. There was also very good agreement in distances among the 3 mixing times. Therefore, spin diffusion effects were minimized. The group of cyclic models with varied backbone carbonyl orientation provided further insight on the structure of the cyclic tetrapeptide IGGN. Our research has shown that it is possible to directly study peptide

35 bond isomerization complementing earlier work which placed more emphasis on the secondary amide peptide bond orientation.

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References 1. Branden, C. Introduction to Protein Structure. Garland. NY. 1998, 89-91. 2. Pauling, L. The Nature of the Chemical Bond. Cornell. NY. 1948, 10-14. 3. Berg, J. Biochemistry. W. H. Freeman. NY. 2006, 37-38. 4. Balci, M. Basic 1H- and 13C- NMR Spectroscopy. Esevier. Boston. 2005, 3-4. 5. Eberhardt, E., Loh, S., Raines, R. Tetrahedron Letters. 1993, 34, 3055-3056. 6. Li, P.S., Chen, E., Shulin, S., Asher, A. J. Am. Chem. Soc. 1997, 119, 1116. 7. Reimer, U., Elmokdad, N., Schutkowski, M., Fischer, G., Biochemistry, 1997, 36, 13802. 8. Scherer, G. J Amer. Chem. Soc. 1998, 120, 5568-5574. 9. Wtrich, Kurt. NMR of Proteins and Nucleic Acids. John Wiley. 1986, 117-120. 10. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., Sykes, B. D., J. Biomol. NMR. 1995, 6, 135-140. 11. Homans, S. A Dictionary of NMR. Clarendon Press. Oxford. 1992, 148-150 12. Goddard, T. D. Sparky 3. University of California, San Francisco. 13. Perrin, C., Gipe, R. J. Am. Chem. Soc. 1984,106, 4036.

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