Current Fluoroorganic Chemistry - American Chemical Society

Chapter 23

Downloaded by UNIV MASSACHUSETTS AMHERST on September 27, 2012 | http://pubs.acs.org Publication Date: January 11, 2007 | doi: 10.1021/bk-2007-0949.ch023

Solvent-Peptide Interactions in Fluoroalcohol-Water Mixtures C. Chatterjee, G. Hovagimyan, and J. T. Gerig* Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106

Mixtures of trifluoroethanol and water were observed to stabi lize certain conformations of peptides and proteins more than 40 years ago. Other fluorinated alcohols are often more potent in this regard. We describe recent results of intermolecular N O E studies of the interaction of hexafluoroisopropanol and other fluoroalcohols with small peptides, including melittin, analogs of angiotensin containing fluorophenylalanine and "trp-cage", a 20-residue peptide designed by N . H . Anderson and co-workers. In many cases, the results provide evidence of strongly preferential interactions of peptides with the fluoroalcohol component of a solvent mixture.

Background The peptide hormone angiotensin II plays a central role in the processes that lead to coronary heart disease and hypertension because it is linked to the regula tion of blood volume, electrolyte balance and vasoconstriction. A precursor to the octapeptide, angiotensiogen, is produced by the liver. The enzyme renin cata-

© 2007 American Chemical Society

In Current Fluoroorganic Chemistry; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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lyzes production of an inactive decapeptide that is the substrate for angiotensinconverting enzyme ( A C E ) which removes two amino acids to produce the physiologically active hormone. Angiotensin II interacts with several classes of G-protein-coupled receptors. Inhibitors of A C E such as Captopril are important drugs for control of hypertension, as are materials that block interaction of an giotensin II with its receptors. Figure 1 summarizes these biochemical events.

Figure 1. Schematic of the renin-angiotensin system (ACE, angiotens inconverting enzyme; ATI, AT2, ATn, various angiotensin 11 receptors). The figure is based on the website www.hypertensiononline.org.

Much effort has been devoted over the last 30 years to attempts to define the conformation or conformations of angiotensin II in solution, in the hope that knowledge of the preferred structure of the octapeptide would guide design of small molecules that would inhibit A C E or be effective in blocking interaction of the hormone with its receptors. Physical studies, including circular dichroism, Raman spectroscopy and N M R spectroscopy, have led to a variety of suggested structures. A recent N M R study of the peptide in aqueous solution at 4°C has supported the conclusion that angiotensin II under these conditions exists pre dominantly as a U-shaped molecule (1). Similar experiments with angiotensin II in phospholipid micelles led to a similar structure (2). Fluorine-containing analogs of angiotensin II have been prepared to probe the interaction of the hormone with its receptor and to explore enhancement of the bioavailability of drugs. Vine and co-workers showed that 4-fluorophenylalanine at position 8 gave a compound equally as potent as the native hormone (3). The same group confirmed an earlier observation by Bumpus and Smedley that 4-fluorophenylalanine at position 4, the tyrosine position, produced an inac tive compound that is an antagonist of native angiotensin II. Replacing valine-5


381


by hexafluorovaline afforded a peptide that was at least as active as the native material but was more resistant to proteolytic degradation (4). Replacement of phenylalanine-8 with pentafluorophenylalanine led to reduced contractile activity and antagonism of the effects of the native material (5).

Figure 2. Solution conformation of angiotensin II proposed by Tzakos, et al. (1).

Several methods are available for introducing fluorine "labels" into peptide and protein structures. One of the reasons for doing this is that fluorine N M R spectroscopic studies of a system are thereby enabled (6). However, it is pres ently not possible to predict how the presence of fluorine will alter local or global structure and dynamics of a biomolecule.

Goals of O u r Work Our goal when starting this work was comparison of the conformations and conformational dynamics of analogs of angiotensin II in which position 4 is oc cupied by phenylalanine, tyrosine or 4-fluorophenylalanine. That is, we hoped to compare compounds that differed only by the presence of a hydrogen atom, a fluorine atom, or a hydroxyl group at the para position of the residue occupying a portion of the octapeptide that appears to be critical for biological activity.

ASPi - ARG - VAL - X4 - VAL - HIS - PRO - PHE 2

3

5

6

?


8


382 Bumpus and collaborators have shown that adding fluorinated alcohols to aqueous solutions of angiotensin II analogs apparently has the effect of stabiliz ing certain conformations (7). We planned to use alcohols such as trifluoroethanol or hexafluoroisopropanol as cosolvents, hoping that this would produce sys tems in which a single peptide conformation was dominant. Conformational studies of peptides by N M R rely on the establishment of constraints that must be satisfied by the deduced structure; the reliability of the structure is dependent on the number and quality of the experimental constraints. Typically, the constraints used are short-range internuclear distances that are estimated by intramolecular nuclear Overhauser effects (NOEs). In an effort to increase the number of con formation-determining constraints available we planned to explore the use of intermolecular NOEs. As is almost always the case in research, things have not developed exactly along the path that was initially envisioned and all that can be done for the present is to describe some observations made as we progress to ward the original goals.

Intermolecular Overhauser Effects The nuclear Overhauser effect (NOE) is the change in the observed intensity of a N M R signal that results when the magnetization associated with another spin is perturbed (8). The N O E is a consequence of nuclear spin-nuclear spin dipole-dipole interactions that contribute to relaxation.

Q Figure 5. Intramolecular dipole-dipole relaxation in a molecule containing spins H andX. Intramolecular NOEs arise when two interacting spin dipoles, say a proton H and some other spin X , both assumed to be spin 1/2 nuclei here, are contained in the same molecule (Figure 3). It is common to imagine that spin H is at the center of a sphere of radius I H X , the distance between the two spins. Nucleus X "skates" on die surface of the sphere as the molecule tumbles in solution, chang ing the orientation of the H - X internuclear vector. The re-orientation of the molecule is described in terms of a rotational correlation time T which is apc


383 proximately the time required for the molecule to rotate in any direction through an angle of about one radian (9). The intramolecular N O E resulting from the H — X interaction depends on T and THXWhen the interacting spins H and X are on different molecules, their dipolar interaction is modulated by mutual diffusion of the two molecules. The simplest description of the interaction in this situation assumes that the spins are con tained within two spherical molecules that have radii r and r , respectively (Figure 4). The closest the two spins can approach each other is r + r = a. The time dependence of the interaction of the spheres is assumed to be described by the mutual diffusion coefficient (D = D + D ) where D and D are the corre sponding translational diffusion coefficients. A correlation time (T) for the inter action of H and X can be taken as the time required to diffuse the distance a with c

H

x


H

H

x

H

x

x

Figure 4. Dipolar interaction between spins H and X when the spins are in different molecules. Typically, H is contained in a solute molecule and X is part oj the solvent molecules. 2

x = a /D (10-12). The cross relaxation rate c^x for the intermolecular interaction of the spins H and X when the signal for spin H is observed is given by

for the experiment and the correlation time T , and N is the concentration of molecules containing spin X (10). The equation for a is based on the assumption that solvent molecules can approach the sphere representing the solute equivalently from all directions. Real solute molecules have a shape and a solvent molecule will make approaches to the solute from different directions that are non-equivalent due to the shape of the solute molecule. (For the present, we continue to regard the solvent molecule as a sphere with spin X located at its center.) Some solvent approach paths will x

H X


384


allow the solute hydrogen and solvent molecule to interact at their van der Waals contact distance. Other approaches will involve interactions at distances longer than this because of steric interference. To take into account the shape of the solute molecule as it interacts with solvent molecules, we used an empirical method that assumes that the contribution to G H X of a single H-solvent spin inter action over a small element of the solute-solvent contact surface can be com puted using the standard equations. Summing the contributions associated with all surface elements is assumed to give the aggregate N O E for a solute hydrogen.

Reliability of Calculated Intermolecular N O E s We have done several tests of the reliability of the computation method de scribed for estimating intermolecular cross relaxation rates. One test system con sisted of 1,3-di-t-butyl benzene dissolved in tetramethylsilane (TMS). This sol ute was chosen because the steric bulk of the t-butyl groups makes solvent ap proaches to proton H2 difficult. T M S was used as the solvent; this molecule is nearly spherical and we believed that this non-polar molecule would likely not exhibit strong interactions with the solute.

1,3-di-t-butylbenzene Cross relaxation rates were compared to those calculated employing the method outlined and experimental values of the diffusion coefficients for solute and solvent in the samples examined. We found reasonably good agreement be tween the experimental cross relaxation terms and those predicted from theory (13). In particular, the "screening" of proton H2 by the adjacent t-butyl groups seemed to be captured correctly by the calculations. Another test was done with the cyclic dipeptide alanyglycyldiketopiperazine dissolved in water (14). Solute proton-water proton NOEs were determined. At low pH, where the complications from exchange of the N - H protons of the solute with the solvent protons are relatively minor, there is again good agreement be tween the observed and calculated intermolecular cross relaxation terms (Table I). The samples examined contained traces of trimethylsilylpropionic acid (TSP) to provide a reference signal; solvent-methyl proton cross relaxation of this ref erence species was also in good agreement with G H H computed by our method.


385


alanylglycyldiketopiperazine

Table I. Solvent-alanylglycyldiketopiperazine intermolecular cross relaxation rates in water (25°) a

Solute proton

NOE

a -73. 29. 24. -100. 29. m

AlaNH Ala C a H Ala C H GlyNH Gly C H 3

2

TSP

3

ln

.]

xlQr s (-67., 20.) (19., 19.) (20., 20.) (-110., 21.) (20., 20.)

19. (20.)

„ROE

ff

m

3

ln

-.

x lOr s nd 22. (25.) 27. (27.) nd 26. (26.) 23. (26.)

a

The first number given in parentheses is the calculated initial cross relaxation rate. Its value depends on intermolecular dipolar interactions with water as well as exchange with protons of water. The second number is the calculated contribution of intermolecular dipolar interactions only. When only a single number is present in parentheses it is the calculated contribution of the intermolecular dipolar interactions. The calculated rotating frame cross relaxation rates includes only the effects of intermolecular dipolar interac tions with water. The rate constants for solvent exchange of alanyl and glycyl N-H pro tons were 0.96 and 0.14 s" , respectively (14). 1

Our original notion was that a solvent-peptide intermolecular N O E could provide structural constraints because its magnitude should depend on the dis tance of closest approach and thus reveal spins of a molecule that are "exposed" to the solvent, as opposed to buried within a structure. Calculated cross relaxa tion terms confirm that side chain protons of a small peptide are generally ex posed to solvent to about the same extent in all conformations. The big differ ences are in the solvent-backbone proton NOEs. In helical conformations pep tide N - H and Coc-H protons are protected from interactions with solvent spins and should exhibit reduced cross relaxation interactions solvent spins.


386

Interactions of Peptides with Fluoroalcohols


Alanylglycyldiketopiperazine. Our first indications of "trouble" ahead came when we determined the proton-fluorine cross relaxation rates for the cyclic dipeptide alanylglycyldiketopiperazine dissolved in aqueous solutions of a vari ety of fluorinated alcohols (15). In every solvent, measured cross relaxation rates exceeded those predicted, sometimes by a substantial amount (Table II). Berger and his coworkers in Leipzig reported similar results about the same time (16, 17). They attributed the enhanced proton-fluorine cross relaxation rates to selective solvation. In effect, the local concentrations of fluoroalcohol near some of the spins of the peptide were assumed to be higher than the concentra tion of the alcohol in the bulk of the solution. Their results were consonant with the observation that trifluoroethanol can accumulate on the surface as well as penetrate to the interior of protein molecules (18).

Table II. ^ { " F } Intermolecular Cross Relaxation Rates of Alanylglycyldiketopiperazine Protons in Fluoroalcohols 8

a

3

H F

J

x 10

s'

50%TFE

cr x HF

3

1

10 s'

50%HFA

G X HF

3

10

s

'

]

35% HFIP

G X HF

3

1

10

s"

80%PTFB

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

12.

5.1

20.

4.7

13.

4.6

49.

9.0

a

TFE, trifluoroethanol; HFA, hexafluoroacetone hydrate; HFIP, hexafluoroisopropanol; PTFB, perfluoro-t-butanol Angiotensin analogs. Figure 5 shows the proton N M R spectrum of phe-4,val-5 angiotensin II in 40% trifluoroethanol/water at 10° as well as a peptide protonsolvent fluorine N O E spectrum. There are strong, positive but variable values of (JHF for the Phe, V a l , Pro, His side chains. The observed cross relaxation rates for some side chain protons are close to the values expected (~3 x 10" s' ) on the basis of the bulk concentration of the trifluoroethanol and the diffusion constants of the peptide and solvent components. Other side chain cross relaxation rates are 3-4 times larger than expected, a result that appears to be consistent with the notion that the local concentration of the fluoroalcohol is larger than the bulk concentration, particularly near the histidine and phenylalanine side chains. Sol vent-solute cross relaxation rates for the peptide N - H or the C a - H protons are 3


1

387


close to zero in this system. That is, there are at best weak contacts between the solvent fluorines and the backbone protons of the octapeptide, a result that indi cates the peptide is predominately in a helical conformation. We have also determined the cross relaxation rates for water protons of the solvent with the solute. These are generally smaller than the predicted values by up to a factor of 2, an observation consistent with the presence of high local con centrations of fluoroalcohol.

L A bw JVJLi

IMLV 10

9

8

7

0

0

4

3

2

1

0

ppm

Figure 5. Lower spectrum: ID proton NMR spectrum of 4-phe,5-val angiotensin II in 40% trifluoroethanol/water at 10° and 500 MHz. Upper spectrum: H{ F} intermolecular NOE spectrum obtained by inversion of the fluorine line of the solvent. The mixing time for the NOE spectrum was 500 ms. l

l9

Melittin. We have done similar experiments with melittin, a 26-residue peptide from bee venom (19). The structure of melittin in 35% hexafluoroisopropanol is a bent helix (Figure 6). The break in helical structure takes place from amino acids gly-12 to leu-16. Intermolecular NOEs were determined between the hexafluoroisopropanol fluorine spins and the solute protons. There are some overlaps in the proton spectrum, so the determination of the G F was not always clean as we would like. However, most side chains show positive solvent NOEs that are close to those predicted by simply assuming diffusive encounters of the peptide and the solvent molecules. Similarly, many N - H protons have NOEs that are close to those preH


388


dieted (Figure 7). However, the backbone N - H protons in the helix-break region show large effects that are in the opposite direction (negative) from those pre dicted.

Figure 6. Representation of the conformation of melittin in 35% hexafluoroisopropanol/water. The drawing represents an overlay of the ten "bestfit" structures determined by analysis of intramolecular ^^H} NOEs (19).

X

Figure 7. Comparisons of observed (dark bars) and calculated (light bars) values of

Current Fluoroorganic Chemistry - American Chemical Society

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