On the low energy solution conformation of somatostatin

Vol. 100, No. 3,198l

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June 16, 1981

1148-1153

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ON THE LOW ENERGY SOLUTION CONFORMATION OF SOMATOSTATIN Byron H. A&on,+

Ralph Hirschmannt William J. Paleveda!? Stephen F. Bradyttand Daniel F. Vebertt Merck Sharp & Dohme Research Laboratories

West Point, Pennsylvania 19486 and Rahway, New Jersey 07065 Received

April

22,1981

SUMMARY A study of the temperature dependence of the chemical shifts of the protons of somatostatin reveals selected upfield shifts consistent with a low energy conformation having features similar to those proposed for the bioactive conformation. Aromatic ring proton assignments were made by a study of somatostatin selectively deuterated on two of the aromatic rings. Improved yields for I2 induced cyclization of the sulfur protected somatostatin precursor were obtained by quenching oxidizing species with zinc dust. Our studies of analogs of somatostatin two structural

have led us to infer the existence of

features of the bioactive conformation

in the solution conformation.

A hydrophobic

not recognized as being present

“stacking”

of the aromatic

Phe-6 and Phe-11 was inferred from the observation of high biological

rings of

activity

in an

analog wherein these two amino acids were replaced by a cystine residue (1). The covalent disulfide bond can replace the aromatic rings while other substitutions preclude bonding result

in reduced potency.

In addition,

chain of lysine-9 to the aromatic ring of tryptophan-8

a proximity

which

of the side

was inferred from the upfield

shift of the y-methylene of lysine in the highly active D-Trp8 analogs. In somatostatin itself

the y-methylene

of lysine does not show the same degree of shielding.

has there been any direct therefore

evidence for the stacking of Phe-6 and Phe-Il.

It was

proposed that the solution and bioactive conformers might be different

tRahway, New Jersey 07065 tbest Point, Pennsylvania 19486 0006-291X/81/111148-06$01.00/0 Copyright All rights

Nor

0 I981 by Academic Press, of reproduction in any form

Inc. reserved.

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Vol. 100, No. 3,1981

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Temperature*

ical shifts of selected CD3OD.

dependence of the chemprotons in somatostatin in

A more recent study of the NMR spectrum of somatostatin recognition

has led to the

that the T-CH2 of lysine 9 was indeed shielded, but to a lesser degree

than seen in D-Trp-8 analogs (3,4).

The resonance, therefore,

lies on the high field

edge of the general aliphatic envelope with an upfield shift of nearly 0.3 ppm relative to the normal in lysine containing evaluate the temperature statin.

peptides.

This observation

has prompted

us to

dependence of the chemical shifts of protons of somato-

These studies provide evidence that the low energy conformation

may be the same as that inferred

for the bioactive

The NMR spectra of somatostatin

conformation.

in methanol-d4

-5O’C to 25’C reveal 3 highly temperature

in solution

over the temperature

range

dependent resonances (Figure 1). Two

of these are recognized as being the ortho and meta protons of one of the three phenylalanines. lysine-9.

The third temperature

Over the temperature

dependent resonance is the y-methylene

of

range studied, a shift of nearly 0.3 ppm is observed

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for each of these, while most other parts of the spectrum remain essentially invariant as exemplified in Figure 1. In order to unequivocally assign the unusually temperature dependent

phenyl ring, a mixture

Pentadeuterophenylalanine

of selectively

was introduced

alanine and pentadeuterophenylalanine nine 7 remained

unlabelled.

deuterated

analogs was prepared.

in position 11; an equal mixture of phenyl-

was introduced in position 6, while phenylala-

The intensities

of the temperature

resonances were reduced 50% in this analog mixture,

thus allowing

dependent

Phe

assignment to

phenylalanine-6.

EXPERIMENTAL NMR spectra were determined at 300 MHz using a Varian SC-300 spectrometer equipped with a Fourier transform accessory. The temperature sensitive signals at 6.80 6 (doublet) and 6.99 6 (triplet) were assigned to the ortho and meta protons respectively in the same aromatic ring based upon multiplic!relatiareas, and double irradiation experiments. The temperature dependence of selected protons is indicated in Figure 1. The data in Figure 1 were obtained at a concentration of 6 x IO-3 M. Nearly identical results were obtained at a concentration of 1 x lo-3M, demonstrating that aggregation is not an important factor contributing to the observed temperature dependent signals. The 50% loss of area of the shielded aromatic protons in the deuterated analog was obvious from a comparison of the spectra in D20 of labelled and unlabelled preparations. Comparison of the spectra in CD30D (Fig. 2) resulted in the same conclusion. 61t was apparent that in the spectrum of the deuterated analog, the area of the Phe -H2,6 signal was approximately one proton relative to Trp H4 or H7. Synthesis of Somatostatin

and Deuterated

Analogs:

a. Bis-Acm-dihydrosomatostatin was prepared by the solid phase method from Boc-Cys(Acm)-O@using a Beckmann 990 peptide synthesizer with a synthesis program essentially as we have previously described (5). Boc amino acids were used throughout. Due to the relative scarcity of pentadeuterophenylalanine (6) reduced excess (ca. 1.5 equivalents) of the Boc derivative was employed (couplings of residues 6 aa II), using activation by DCCI in the presence of I-hydroxybenzotriazole hydrate with DMF as solvent. A single treatment for 19 h was sufficient to achieve complete coupling, as judged by testing with ninhydrin reagent (7). Removal of the peptide from the solid support was accomplished with liquid HF in the presence of anisole as scavenger. The sulfur-protected 14-peptide was isolated after purification by gel filtration in 2 M acetic acid (Sephadex G-25 superfine), in 40% yield based on polymer-bound Boc-Acm-Cys. The product was identical to material prepared by solution methods (8). b. Somatostatin was prepared directly from the bis-Acm dihydroderivative by oxidative cyclization using iodine in 98% acetic acid
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6.6

Somotostatin: Aromatic Region (CDsOD) 300 MHz Upper Trace: Lower Trace:

Normal Spectrum PIE’50% 0 PM’0% D PM”tOO% D Figure

2

the appearance of such tryptophan-modified by-products during the process of isolation, leading us to believe that species of active halogen can persist, even after treatment with thiasulfate. Zinc treatment, by both destroying excess iodine and reducing by-product formation, has resulted in significantly improved yields. After filtration to remove zinc, the solution was concentrated in -- vacua. The somatostatin obtained was purified by gel filtration with Sephadex G-25 superfine in 50% acetic acid, with an isolated yield of 42% based upon the bis-Acm derivative. The yield and quality were comparable to that obtained by other methods. Amino acid analysis after acid hydrolysis: Lys 2.02, Asp 1.01, Thr 2.04, Ser 0.93, Gly 0.98, Ala 0.99, Phe 3.04. UV (water) X max 288 (40501, 280 (46401, 273 (4430) Analytical reversed phase HPY): 97%0pure. TLC (Whatman Kl): Rf 0.46 ?EbAc: Pyr: HOAc: H20) 5:5:1:3. [a] I, -33.3 (C 0.2, 1% HOAc). DISCUSSION The marked temperature

dependence of the chemical shift of specific protons

supplies evidence that the lowest energy conformation

of somatostatin

of lysine in the shielding cone of an aromatic ring and simultaneously meta protons of phenylalanine-6

has the y-CH2 the ortho and

also lie in the shielding cone of an aromatic ring.

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(An aromatic ring is ihe only type of anisotropic group large enough to simultaneously shift

both the ortho and meta protons.)

which influences D-tryptophan

the y-CH2 of lysine is the indole of tryptophan,

containing

analogs.

one of the other phenylalanine Phe’s 6 and II.

It is most likely that the aromatic

ring

as is the case in

The ring protons of Phe-6 must be shielded by residues, consistent

The fact that only one aromatic

the ring stacking be perpendicular

with the proposed stacking of ring is so shielded requires that

rather than parallel.

Previous theoretical

studies

have pictured low energy stacking as being parallel (91. However, crystal structure studies of benzene (IO) and phenylalanine (II) show perpendicular

stacking of phenyl

rings. Indeed, ab -- initio calculations carried out in these laboratories (12) demonstrate stabilization

of perpendicular

stacking only.

Thus NMR studies now reveal structural conformation activity

which are consistent

features in the low energy solution

with structural

features inferred from structure

studies as being present in the receptor bound, “bioactive”

These same structural

conformation .

features which do not dominate at 25OC in the natural hormone

are more prevalent in the more biologically do not exclude a further conformational

potent

D-Trp analogs (2).

change to the bioactive

Our studies

form after receptor

bindings as proposed by Momany (13). In the absence of evidence for such a change, we prefer to view the now demonstrated same as the bioactive

low energy solution conformation

as the

form.

REFERENCES 1. 2. 3. 4. 5. 6.

Veber, . F., Holly, F. W., Paleveda, W. J., Nutt, R. F., Bergstrand, S. J., Torchiana, M., Glitzer, M. S., Saperstein, R., Hirschmann, R. (1979) Proc. Natl. Acad. Sci. U.S.A. B, 2636-2640. Arison, B. H., Hirschmann, R., and Veber, D. F. (1978) Biorg. Chem., l, 447-451. Hallenga, K., Van Binst, G., Scat-so, A., Michel, A., Kroppenberg, M., Dremier, C., Brison, J., and Dirby, J. (1980) FEBS Letters, 2, 47-52. Buffington, L., Garsky, V., Massiot, G., Rivier, J., and Gibbons, W. A. (19801 Biochem. Biophys. Res. Comm., z, 376-384. Strachan, R. G., Paleveda, W. J., Bergstrand, S. J., Nutt, R. F., Holly, F. W., and Veber, D. F. (1979) J. Med. Chem., 2, 586. Pentadeutero phenylalanine was prepared by A. Skyerenina of Merck Sharp & Dohme Canada Limited.

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Kaiser, E., Colescott, R., Bossinger, C., and Cook, P. (1970) Anal. Biochem. 2, 595-598. Veber, D. F. in “Proceedings of the 4th American Peptide Symposium,” J. Meienhofer and R. Walter (Eds.), pp. 307-316, Ann Arbor Publishers, 1975. Nemethy, G. and Scheraga, H. A. (1962) J. Phys. Chem. 66, 1773-1789. Caillet, J. and Claverie, P., Acta Cryst. A31 448-461 (1973. Al-Karaghouli, A. R. and Koetzle, T. F.3eta Cryst. B3J, 2461-2465 (1975). Thomas, K. A., Thomas, T. B., Smith, G. M., and Feldman, R. J. (manuscript in preparation). Momany, F. A. (1980) B&hem. Biophys. Res. Comm. z, 61-66.

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