THE JOURNAI. OF ~I”I.“GICAL CHEMISTKY Vol. 250, No. 5, Issue of March 10, pp. 1912 -1918, 1975

Printed in U.S.A.

The Synthesis of Neurotensin* (Received for publication, June 3, 1974)

ROBERT CARRAWAY~ ANn SUSAN E. LEEMAN

From the Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 021 I.5

A tridecapeptide having the amino acid sequence, ~(;lu-I,eu-‘l’yr-Glu-Asn-I,ys-Pro-Arg-Pro-’~yr- Ile-Leu-OH, (The nomenclature and symbols follow the suggestions of the IIJPAC-KJH Commission on Biochemical Nomenclature ((1972) J. Rio/. Chem. 247, 977).) has heen synthesized by the Merrif’ield solid-phase procedure. The synthetic scheme chosen involved synthesis of the peptide in the (Gln) fhrm and cyclization to the ( <Glu) form. After purification, the (Gin) peptide was obtained in a 7% yield and the ( <Glu) peptide was ohtained in a W%’ yield. The ( <Glu) peptide was found to he chemically and biologically indistinguishable from the tridecapeptide, neurotensin, recently isolated from hovine hypothalami.

The discovery and isolation of a new vasoactive peptide, neurotensin, from bovine hypothalami has recently been re- ported hy this laboratory (1). In addition to the biological properties that classif’y it as a kinin, neurotensin can also cause a rapid increase in plasma glucose levels. Structural studies on the isolated tridecapeptide have indicated that neurotensin is composed of L-amino acids in the sequence <Glu-Leu-Tyr- Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH (2). This paper describes the synthesis of a peptide with the above structure hy the Merrifield solid phase technique (Z) and presents data demonstrating that the purified synthetic product is chemi- cally and biologically indistinguishable from the isolated hypothalamic substance.

EXPEHIMENTAI. PHOCEDUKE

The manual system for solid phase peptide synthesis (4) was obtained from Schwarz-Mann. The chloromethylated resin, copoly- styrene-2%divinylbene, with 2.7 mmol of chloride per g was a generous gift from Dr. Arnold Marglin, Department of Protein Chemis- try, Tufts University School of Medicine, Boston, Mass. BOC’-amino acids, BOC-amino acid p-nitrophenyl esters, and N,N’-dicyclohexyl- carbodiimide were obtained from Schwarz-Mann. An assessment of the optical purity of the BOC-tyrosine preparation obtained from Schwarz-Mann by the procedure of Manning and Moore (5) revealed that the material was >99% I. configuration. Pyridine hydrochloride was made by bubbling dry HCl gas through a solution of pyridine in ether and drying the resultant crystalline precipitate. N,N’-dimethyl- formamide was purified by passage through a column of 4 A molecular sieves (Matheson, Coleman and Bell) and then stored a few days over these sieves prior to use (6). Anhydrous trifluoroacetic acid and

*This research was supported in part by National Institutes of Health Grant 7 ROl AM 16510.01.

$ This work is part of a dissertation submitted to Brandeis Univer- sity, Waltham, Massachusetts in partial fulfillment of the require- ments for the degree of Doctor of Philosophy. Predoctoral Trainee supported by National Institutes of Health Training Grant GM-212.

‘The abbreviations used are: BOC, t-butoxycarbonyl; Dns, 1- dimethylaminonaphthalene-5.sulfonyl.

triethylamine were reagent grade products from Eastman and all other solvents used were Fisher-certified. Sephadex media, enzymes, and animals were obtained as previously stated (1, 2).

Methods

The general procedures of the manual solid phase method were followed (3). The synthetic scheme chosen (Fig. 1) involved synthesis of the protected tridecapeptide in the (Gin) form (Structure I), cleav- age from the resin to yield Structure II, removal of protecting groups to give Structure III, and cyclization of the product to the (<Glu) form (Structure IV). Partial cyclization was found to occur during cleavage, deprotection, and purification and both tridecapeptides (Structures III and IV) were obtained as products.

Five grams of chloromethylated resin (2.7 mmol of chloride per g) were esterified with BOC-Leu by the method of Marglin (6). Four grams of the resulting BOC-Leu-resin were placed in large reaction vessel of the manual type and the appropriate BOC-amino acids were successively coupled to the NH, terminus of the growing peptide chain. Most of the couplings were mediated with dicyclohexylcar- hodiimide, using a %fold excess of HOC-amino acid and dicyclohexyl- carhodiimide in CH,Cl, and a reaction time of 2 hours. BOC-Arg(N0,) was dissolved in a minimum amount of dimethylformamide and diluted with CH,CI,. BOC-asparagine and BOC-glutamine were coupled as the active p-nitrophenyl esters using a h-fold excess of reagent for 20 hours (7). The side chain protecting groups used were Glu (OBzl), Tyr (Bzl), 1,~s (Z), and Arg (NO,). Each cycle of coupling was performed essentially as described hy Gutte and Merrifield (8), except that 25%, instead of 50%~ (v/v) trifluoroacetic acid-CHzCl, was used and the reaction time was increased to 30 min.

The completeness of the deprotection and coupling reactions was quantitatively assessed by monitoring the amount of free amine on the entire batch of peptide resin (9). The completeness of coupling was also qualitatively assessed by the ninhydrin method (IO). The course of the synthesis was followed by examining the homogeneity of the growing peptide after the chain reached 6, 8, 10, and 12 residues in length. At each of these stages, peptide was cleaved from 5% to 150.mg samples of the resin, deprotected, and examined by high voltage paper electropho- resis. Amino acid analyses were performed before and after paper electrophoretic purification of these fragments of neurotensin. The protected peptides were cleaved from the resin using HBr-trifluoro- acetic acid (11). The HBr gas was purified by passage through solutions of resorcinol and anisole was added to the suspension of peptide resin. Catalytic hydrogenation to remove the NO? groups from the 2

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resin and protected amino acids

Merrifleld Synthesis

DCC/CH2C12

Bzl OBzl

Cleavage HBr/TFA

NO2 NO2

I I H-Gln-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile -Leu-OH

(11)

I Deprotection

1

H2/Pallad~um

H-Gln-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile -Leu-OH

I

(III)

Cyclization

1

Heat/Acid

<Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile -Leu-OH (IV)

FIG. 1. Diagrammatic representation of the pathway chosen for the synthesis of neurotensin. DCC, dicyclohexylcarbodiimide; TFA, tri- fluoroacetic acid.

nitroarginyl residues was performed in a Paar hydrogenator using 5% palladium on barium sulfate as catalyst (12). Peptide, at a concentra- tion of 10 mg per ml in methanol-acetic acid-water (lO:l:l, v/v), was shaken with an equal weight of catalyst for 72 hours under hydrogen at a pressure of 6 atm. The mixture was then filtered through Whatman No. 1 paper and the filtrate was rotary evaporated, the residue was dissolved in water, and the solution was lyophilized. An aliquot was removed for acid hydrolysis and amino acid analysis. Yields for the synthesis were calculated from the amount of titrable leucine originally esterified to the resin.

Analytical procedures, enzymic digestions, and biological testings were done as previously described (1, 2). For hydrolysis of peptide resins, 10 to 30 mg f 5% of resin were added to 20 ml of dioxane-12 N

HCl, lOMa M phenol (l:l, v/v) in a round bottom flask and refluxed 18 to 24 hours using an air condenser. The solvent was removed by rotary evaporation and the residue was dissolved in 0.01 N HCl or 0.2 N

sodium citrate buffer, pH 2.2, for application to an amino acid analyzer. Samples of free peptides (20 to 200 nmol) were routinely hydrolyzed with 0.5 ml of constant boiling HCl, lo-’ M phenol in evacuated, sealed tubes at 145” rt 2” for 4 hours (13). The hydrolysis solvent was removed in vacuum in a desiccator containing sodium hydroxide pellets and sulfuric acid. In experiments comparing the hyperglycemic effect of native and synthetic neurotensin, male rats (Charles River Breeding Laboratories) weighing 130 to 150 g were anesthetized with Nembutal, 45 mg per kg, by intraperitoneal injec- tion. Each animal was killed 15 min after intravenous injection of peptide samples and plasma glucose levels measured by the method of Ceriotti and Frank (14).

Chromatography on Sephadex LH-go--Because it was found not to be soluble in water, the crude synthetic material was initially fractioned on Sephadex LH-20 using methanol-water (3:l) as eluant. The lyophilized product after hydrogenation was dissolved in 40 ml of methanol-water (3:1), filtered through Whatman No. 1 paper, and applied in two batches to a 600-ml column of Sephadex LH-20 equilibrated with this solvent. The peptide concentration of the eluates was monitored at 276 nm. Although only one major peak was observed in the column profile (Fig. 2a), paper electrophoresis of aliquots of the column eluates indicated the presence of two major peptides within that peak (Fig. 26). Therefore, the material eluted in Region I was pooled, lyophilized, and further fractionated.

Cation Exchange Chromatography at pH 5.5-The combined mater- ials of Region I (Fig. 2) from two columns of Sephadex LH-20 was dissolved in 150 ml of 0.05 M pyridine acetate, pH 5.5, and applied to a 20-ml column of sulfoethyl-Sephadex equilibrated with this buffer. The column was developed with a linear gradient which was generated using 500 ml of 0.05 M pyridine acetate, pH 5.5, in the mixing chamber and 500 ml of 1.0 M pyridine acetate in the reservoir. The absorbance of the eluates at 300 nm was measured (Fig. 3a) and aliquots of the eluates were subjected to paper electrophoresis and staining (Fig. 3b). The materials in Region I and Region II were pooled separately and samples removed for amino acid analyses, NH*-terminal analyses, and paper electrophoretic analyses. The material recovered from Region I proved to be homogeneous and was designated as peptide NTI. The material in Region II, which appeared to contain a minor contaminant, was pooled, lyophilized, and further fractionated.

Cation Exchange Chromatography at pH 3.2-The material from Region II (Fig. 3~) was dissolved in 150 ml of 0.20 M pyridine acetate, pH 3.1, and applied to a 20-ml column of sulfoethyl-Sephadex which was then developed with a linear gradient made from 500 ml of 0.20 M pyridine acetate, pH 3.1, and 500 ml of 1.5 M pyridine acetate, pH 5.5. The absorbance of the eluates was measured at 300 nm (Fig. 4~) and aliquots were subjected to paper electrophoresis and staining (Fig. 4b). Region I and Region II were pooled separately and aliquots removed for amino acid analyses, NH*-terminal analyses, and paper electropho- retie analyses. The material recovered from Region I was shown to be more of the pure peptide NT,. Region II also contained a homogeneous peptide which was designated as peptide NT,.

Cyclization in 4 N Acetic Acid-Cyclization of the (Gin) tridecapep- tide was affected by treating 1.0 pmol of the NT? peptide with 2.0 ml of 4 N acetic acid overnight at 40”. The sample was lyophilized and subjected to paper electrophoresis at pH 6.5; ninhydrin staining indicated the presence of two peptides running adjacent to peptides

I (a)

1 1.0

’ 2 0.8

2 0.6

z 0.4

E 1 0.2

NT, -MASP= 0.18

NT2-M ASP = 0.36

FIG. 2. a, gel chromatography of crude synthetic tridecapeptide on Sephadex LH-20. Sample, hydrogenated synthetic material represent- ing 1.75 g of resin, estimated to be 0.5 mmol of peptide by its absorbance at 276 nm; column size, 3.8 x 53 cm (resin volume 600 ml); fraction size, 10 ml; eluent, methanol-water (3:l). The void volume (V,) was determined with blue dextran and the column volume (V,) with glucose. Region I refers to Fractions 24 to 34. b, drawing of the ninhydrin stains obtained after paper electrophoresis of aliquots of fractions obtained from (a). Approximately 5 ~1 of each sample were applied at the origin as indicated along with the standard amino acids. lysine (Lys) and leucine (Leu). Conditions: pH 6.5, 86 volts per cm, 36 min. 0, major stain; 0 , minor stain. NT, and NT, refer to the peptides having electrophoretic mobilities relative to aspartic acid (MAsp) of 0.18 and 0.36, respectively.

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(a) regions adjacent to the ninhydrin stains of the spotted standards, NT, and NT,, were acid-hydrolyzed and analyzed for amino acids.

0.4

E' 0.3

i 8 0.2

0.1

(b)

IO 20 30 40 50 60 Fraction Number

NT, -MASP =0.18 NT, -MASp = 0 36

LYS

Leu

0.4

0.3

0.2

01

FIG. 3. a, ion exchange chromatography on sulfoethyl-Sephadex C-25. Sample, material from Region I of Fig. 2a, representing 3.5 g of resin and estimated to contain 0.85 mmol of peptide by its absorbance at 276 nm; column size, 1.2 x 16 cm (resin volume 18 ml); fraction size, 10 ml; column buffer, 0.05 M pyridine-acetate, pH 5.5. ---, an estimate of the pyridine concentration. Region I and Region II refer to Fractions 15 to 30 and 32 to 44, respectively. b, drawing of the ninhydrin stains obtained after paper electrophoresis of aliquots of fractions obtained from (a). Approximately 10 pl of each sample were applied at the origin as indicated along with the standard amino acids, lysine (Lys) and leucine (Leu). Conditions: pH 6.5, 86 volts per cm, 36 min. 0, major stain; C, minor stain. NT, and NT, refer to the peptides observed having electrophoretic mobilities relative to aspartic acid (iWAsP) of 0.18 and 0.36, respectively.

NT, and NT, spotted as standards. The corresponding regions were eluted and aliquots were taken for amino acid analyses and NH&ermi- nal analyses. Samples of the eluate of the NT, region also were subjected to high voltage paper electrophoresis and staining at pH 1.9, pH 3.5, and pH 8.9, with NT, peptide spotted as a standard. Chymotryptic, tryptic, and papain digests of samples of this eluate were also subjected to electrophoresis at pH 3.5 and the relative mobilities of the stained peptide fragments were noted.

Cyclization in Acid-Acetone-To examine the effects of the solvent used to extract neurotensin from hypothalamic tissue (1) on the (Gin) tridecapeptide, 25 nmol of NT, peptide were added to 50 ml of acetone-O.024 N HCl (80:20, v/v) or to 12.5 ml of a hypothalamic extract containing 2.5 g of bovine hypothalamic tissue homogenized in 10 ml of acetone-l N HCl (100:3, v/v). Both solutions were stirred overnight at 4”, centrifuged 15 min at 20,000 x g (Sorvall), and the supematant fractions were set aside. The precipitant fractions were re-extracted with 5.0 ml of acetone-O.01 N HCl (80:20, v/v) for 2 hours at 4” and centrifuged 15 min at 20,000 x g. The pooled supernatant fractions were extracted four times with an equal volume of petroleum ether (bp 36” to 50.9”), evaporated to dryness, the residue was dissolved in water, and the solution was lyophilized. The dried materials were taken up in buffer and subjected to high voltage paper electrophoresis at pH 6.5; the standard peptides, NT, and NT,, were spotted on guide strips. Regions of the paper (2.0 cm in width) were eluted and, after lyophilization, samples were diluted into 0.85% NaCl solution (saline) and assayed for hypotensive activity (1); both peptides, NT, and NT, are active in this assay. The eluates from the

RESULTS

Synthesis-Amino acid analyses of dioxane-HCl hydroly- sates of the esterified resin indicated that 0.53 mmol of BOC-Leu was associated per g of resin. However, quantitative determination of resin-bound amino groups by chloride titra- tion after treatment with 25% trifluoroacetic acid-CH& gave the result, 0.40 mmol of amine (leucine) per g of resin. The difference represents the sum of ion-exchanged leucine and covalently attached but unreactive leucine; only the titrable leucine was considered available for peptide synthesis.

Resin-bound amine was determined through the first four coupling steps of the synthesis and indicated that coupling and deblocking efficiencies were 98 to 100%. As expected, the amount of titrable amine was found to oscillate from values approaching 0 mmol per g immediately after coupling to values around 0.40 mmol per g after deblocking.

Chemical and electrophoretic examination of the growing peptide at the 6-, 8-, lo-, and 12-residue stages indicated that the synthesis was progressing as expected. The amino acid compositions and the NH*-terminal amino acids of the prod- ucts were as expected, and the peptides appeared to be homogeneous by electrophoresis at pH 1.9, 3.5, 6.5 and 8.9.

(0) 1 Liter Gradient

0.2M PyrAc pH3 to 1.5 M PyrAc pH 5.5 H

(b)

1

IO 20 30 40 50 Fraction Number

I

NT, -MASp: 0 I8

NTL-MASP= 037

FIG. 4. a, ion exchange chromatography on sulfoethyl-Sephadex C-25. Sample, material from Region II of Fig. 3a, representing 3.5 g of resin and estimated to contain 0.31 mmol of peptide by amino acid analysis; column size, 1.2 x 17 cm (resin volume 20 ml); fraction size, 10 ml; column buffer, 0.2 M pyridine-acetate, pH 3.1. - - -, an estimate of the pyridine concentration. Region I and Region II refer to Fractions 4 to 18 and 25 to 36, respectively. b, drawing of the ninhydrin stains obtained after paper electropboresis of aliquots of fractions obtained from (a). Approximately 20 ~1 of each sample were applied at the origin as indicated along with the standard amino acids, lysine (Lys) and isoleucine (Zle). Conditions: pH 6.5, 86 volts per cm, 36 min. 0, major stain; <>, minor stain, NT, and NT2 refer to the peptides observed having electrophoretic mobilities relative to aspartic acid (MAsP) of 0.18 and 0.37, respectively.

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At the completion of the synthesis, the peptide-resin (origi- nally 3.5 g) weighed 5.9 g, consistent with the presence of 1.2 mmol (85% yield) of the protected tridecapepeptide on the resin. Following cleavage from the support and hydrogenation, the amino acid content of the product indicated that 1.0 mmol (70% yield) of the peptide was recovered.

Purification and Yield-Although the intention was to cyclize the (Gln) product to the ( <Glu) form after purification, the results show that when subjected to the highly acidic conditions used for cleavage and deprotection a portion of the (Gln) tridecapeptide cyclized. Thus, two peptides, NT1 and NT2, were observed when the crude synthetic material was chromatographed on Sephadex LH-20 (Fig. 2a) and aliquots of the eluates were subjected to paper electrophoresis and stain- ing (Fig. 26). NT1 was adequately separated from NT2 using sulfoethyl-Sephadex (Fig. 3, a and b) and was obtained in pure form. NT, was freed of a minor contaminant by rechromatogra- phy on sulfoethyl-Sephadex, the results of which (Fig. 4, a and 6) also illustrate the conversion of NT* to NT1 that occurs under these conditions. Amino acid analyses of material from Region I (Figs. 3 and 4) and Region II (Fig. 4) indicated that both NT1 and NT, contain the expected 13 amino acids in the appropriate molar ratios. These materials also appeared homo- geneous during electrophoresis at pH 1.9, 3.5, 6.5, and 8.9. The over-all yield of pure tridecapeptide was 42%; 35% was re- covered as NT1 and 7% as NT2.

Structural Studies on NTI-NT1 was shown to be Product IV (Fig. 1). The peptide was shown to lack a free NH2 terminus by reaction with Dns-chloride; however, digestion with pyrroli- donecarboxylyl peptidase was found to generate a new peptide, separable by electrophoresis at pH 6.5 (mobility relative to lysine, 0.35) which possesses an NH*-terminal leucine. Amino acid analysis of a total enzymic digest of NT1 gave Lys(l.O), Arg(2.0), Asn(OS), Glu(0.8), Pro(l.S), Ile(l.l), Leu(2.2), and Tyr(l.8) (free <Glu does not react with ninhydrin). Further sequence work performed on its papain-generated fragments (Table II) established the proper structure.

Structural Studies on NT2-NT2 was shown to be Product III (Fig. 1). Reaction of the peptide with Dns-chloride indi- cated the presence of only NH,-terminal glutamine or glutamic acid. A total enzymic digest performed on 90 pg of material yielded 75 pg of amino acids: Lys(l.O), Arg(l.8), Asn and Gln(2.1), Glu(0.8), Pro(l.8), Ile(l.O), Leu(B.O), and Tyr(l.8). Furthermore, cyclization of the NH,-terminal glutsn ine of peptide NT2 by treatment with 4 N acetic acid under conditions reported to cyclize NHz-terminal glutaminyl residues (15) was found to convert peptide NT* into peptide NTI. After electro- phoresis of the treated sample of NT2 (1 Mmol) at pH 6.5, the ( < Glu) tridecapeptide was retrieved in 70% yield and shown to be indistinguishable from peptide NT1 by the following crite- ria: amino acid analysis, NH,-terminal analysis by reaction with Dns-chloride before and after treatment with pyr- rolidonecarboxylyl peptidase, electrophoretic mobility in four systems, and peptide mappings of tryptic, chymotryptic, and papain-generated fragments.

Identity of NT, to Isolated Native Neurotensin-The syn- thetic ( <Glu) tridecapeptide (NT1) was found to be indistin- guishable from isolated native neurotensin by the following criteria: (a) amino acid compositions of acid hydrolysates of the two peptides were the same (1); (b) attempts at total enzymic digestion of NT1 by several methods gave results similar to those reported for neurotensin (2); (c) NH&erminal analyses using pyrrolidonecarboxylyl peptidase and Dns-chlo-

ride demonstrated the presence of an NH,-terminal pyroglu- tamyl residue in each molecule; (d) ultraviolet spectral analyses gave molar absorption coefficients at 276 nm of 3.0 x lo3 M-’

cm-‘, and 2.8 x lo3 M-’ cm-’ for native and synthetic material, respectively; (e) chromatographic and electropho- retie behavior of the two peptides were the same (Table I); (f) peptide mappings of the tryptic and chymotryptic fragments of the two peptides gave similar results (Fig. 5); (g) amino acid

TABLE I Comparison of chromatographic and electrophoretic behavior of native

and synthetic neurotensin Gel chromatography on Sephadex G-25 was performed in 0.1 M

acetic acid as eluant. Pyridine acetate buffers were used in ion exchange chromatography. The electrophoretic mobilities are ex- pressed relative to an appropriate standard and were calculated by averaging the results obtained from at least three determinations.

System Property Value

Native Synthetic

Sephadex G-25 Sulfoethyl-Sephadex

Paper electrophoresis pH 1.9 pH 3.5 pH 6.5 pH 8.9

Klva Elution molarity

at pH 5.5 Relative mobility

M,,b M,ysC M a,,” MY,’

0.30 0.10

1.00 0.95 0.38 0.37 0.19 0.18 0.28 0.27

0.30 0.10

“K., = elution volume (~3 - void volume (u&/column volume (a,) - void volume (u,,).

“M%r = mobility relative to serine. c Mm = mobility relative to lysine. ’ Ma,, = mobility relative to aspartic acid.

c-2

NT c-3

(0) NINHYDRIN

ORIGIN

C-l

(b) Ct4~0ff INE, o-TOLIDIM

FIG. 5. Stains obtained after high voltage paper electrophoresis of native and synthetic neurotensin and their tryptic and chymotryptic fragments. Twenty nanomoles of native or synthetic neurotensin were digested at a 1: 100 molar ratio of trypsin or chymotrypsin overnight in 0.2 M ammonium bicarbonate, pH 8.0. After lyophilization, the samples were dissolved in 25 ~1 of 0.1 M pyridine-acetate, pH 6.5. The numbers refer to the samples spotted at the origin: 1, synthetic neurotensin (from Region I, Fig. 3a); 2, native neurotensin (isolated from bovine hypothalami); 3, chymotryptic digest of synthetic neuro- tensin; 4, tryptic digest of native neurotensin; 5 and 6, tryptic digest of synthetic neurotensin; 7, synthetic neurotensin; 8, native neurotensin; 9, chymotryptic digest of synthetic neurotensin; 10, chymotryptic digest of native neurotensin. Conditions: pH 3.5, 86 volts per cm, 32 min (migration distance observed for lysine (LYS), 29 cm, and for isoleucine (ILE), 5 cm). After electrophoresis the paper was cut in half and (a) was stained with ninhydrin-cadmium acetate and (b) was stained by the chlorine, o-toluidine method. The spots obtained are labeled as follows: NT, neurotensin; C-l, C-2, C-3, chymotryptic fragments; T-l, T-2, tryptic fragments. Note that the C-l peptide does not stain with ninhydrin.

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compositions of the isolated fragments obtained after cleavage of synthetic neurotensin by trypsin, chymotrypsin, or papain (Table II) were identical with those obtained after cleavage of native neurotensin by these enzymes (2). In addition, sequence analyses performed on the papain-generated fragments (Table II and Ref. 2) give comparable results; (h) kinetic data of the digestion of the two peptides with carboxypeptidase A and B are indistinguishable (Fig. 6); (i) native and synthetic peptides were found to be equipotent in regard to their hypotensive (Table III) and hyperglycemic (Fig. 7) activities in uiuo and their effects on two smooth muscle preparations in vitro (Fig. 8).

Conversion of NT, to NT, by Treatment with Acid- Acetone-When pure NT1 peptide was put through the extrac- tion procedure used to obtain neurotensin from bovine hypo- thalami and the retrieved material was examined for the presence of the NT1 and NT, peptides, it was found that the NT1 peptide, whether in the presence or absence of hypotha- lamic tissue, was quantitatively converted to the NT1 peptide; more than 95% of the recovered peptide (40 to 50% yields) was in the (<Glu) form.

DISCUSSION

These studies serve to confirm the established primary structure of isolated native neurotensin and provide a method for obtaining large quantities of this peptide in pure form. The synthesis described here yielded pure material equivalent to the extraction of 10,000 hypothalamic preparations of 45 kg each.

The synthetic scheme employed (Fig. 1) put to use the tendency of NHa-terminal glutaminyl peptides to cyclize to their pyroglutamyl derivatives (16). This tendency is acceler- ated by heating under acidic conditions (17), and also by exposure to the hydrogen form of a sulfonic acid ion exchanger (18). Thus, both incubation in 4 N acetic acid at 40’ and chromatography on sulfoethyl-Sephadex (Figs. 3 and 4) pro- moted the conversion of peptide NTa to peptide NT1. Appar-

(b)

I I I I 4 I I 10 20 30 40 50 60 70 60 200

Time (Min.1

FIG. 6. a, kinetics of the digestion of synthetic neurotensin with carboxypeptidase A. One hundred nanomoles of the NT, peptide from Region I, Fig. 3 was incubated at 38” with 50 pg of carboxypeptidase A in 0.2 ml of 0.2 M ammonium bicarbonate, pH 8.0. b, kinetics of the digestion of native neurotensin with carboxypeptidase A. Twenty nanomoles of pure peptide isolated from bovine hypothalami was incubated at 38” with 10 fig of carboxypeptidase A in 0.2 ml of 0.2 M ammonium bicarbonate, pH 8.0. Aliquots were withdrawn at the times indicated and the reactions stopped by addition of glacial acetic acid. The lyophilized residues were subjected to amino acid analysis.

TABLE II Molar ratios of amino acids in isolated fragments obtained after cleavage of synthetic neurotensin by trypsin, chymotrypsin, or papain; sequence

analyses of papain-generated fragments A 300- to 500~nmol sample of the NT, peptide was digested with trypsin, chymotrypsin, or papain as described under “Methods.” After paper

electrophoresis, the peptide fragments were obtained in the following yields: T-l, 70%; T-2, 60%; C-l, 70%; C-2, 80%; C-3, 65%; P-l, 35%; P-2, 65%; P-3, 50%. (1) <Glu established by treatment with pyrrolidonecarboxylyl peptidase; (-) sequence established by the dansyl method; and (7)+equence established with carboxypeptidase A or B.

Peptide fragment” <Glu - Leu - Tp _ Glu - Asn - Lys - Pro - Arg - Arg - Pro - Tyr - Ile - Leu

T-l l.OJ 1.0 1.0 1.0 1.0 1.0 1.0 1.0

T-2

C-l 1.01 1.0 1.0

c-2 1.0

c-3 I

1.0

1.0 1.0 1.0 0.9 0.9

.O 1.0 1.0 1.0 1.0 1.0

1.0 1.0

P-lb 1.01 02 @ g

P-2 if0 G 1.0 g

P-3 i2 li? 1.0 02 02

a Fragments are lettered according to the enzyme used in their generation (T, trypsin, C, chymotrypsin, P, papain) and numbered from the NH2 terminus.

b Another minor product of papain digestion (5 to 20% yield) is the C-l peptide.

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TABLE III Comparison of effect of native and synthetic neurotensin on systemic

blood pressure of anesthetized rats

a Fall in carotid blood pressure (mean + S.E.) measured 30 s after injection of a test sample into the femoral vein of rats weighing 180 to 250 g and having a starting blood pressure between 120 and 160 mm Hg. Each injection was done in a separate rat because both prepara- tions exhibit acute tachyphylaxis. In parentheses are the number of animals per group. Experimental procedure was as previously de- scribed (1).

b Highly purified neurotensin from bovine hypothalami (biological

Dose of peptide

c-t lmin

Guineo Pig Ileum Rat Duodenum

FIG. 8. Comparison of the effects of native neurotensin and syn- thetic neurotensin (NT,) on the contractility of freshly dissected smooth muscle preparations. At the upper left, the isotonic contrac- tions of guinea pig ileum caused by addition of varying amounts of native neurotensin to the bath as indicated at the arrows and below the responses to equal amounts of NT,. At the upper right, the relaxation of rat duodenum caused by adding varying amounts of native neurotensin and below, the responses to equal amounts of NT,. On the abscissa, time in min and at the arrows, the final molar concentra- tions of peptides.

specific activity approximately 1000 doses per mg). c Material from Region I (Fig. 3a).

IJ NATIVE NEIJROTENSIN _ 150

z t t?d SYNTHEYIC NEUROTENSIN

1.0

DOSE OF NEUROTENSIN (nmoles/kg )

FIG. 7. Comparison of the hyperglycemic effect of native and synthetic neurotensin. Plotted is the increment in plasma glucose levels (mean + SE) in each test group (number of animals given in parentheses) above the saline-injected control group, eight animals with a mean plasma glucose level of 160 + 8 mg/lOO ml. Experimental procedure is described under “Methods.” The native material used was highly purified neurotensin from bovine hypothalami (biological specific activity approximately 1000 doses per mg) and the synthetic material was from Region I (Fig. 3a).

ently, the acidic conditions used for cleavage of the synthetic peptide from the resin and for its deprotection were also sufficient to bring about a significant amount of cyclization. Because the NT1 and NT2 peptides are easily separated and purified, this approach permits one to obtain both the glutami- nyl and the pyroglutamyl tridecapeptides in homogeneous form. The over-all yield of pure peptide obtained from this synthesis, 42%, is comparable to the yields reported for a number of similar peptides made by the solid phase procedure (19-21).

The question of the native state of neurotensin is still unanswered. Our results do not exclude the possibility that the native molecule exists in situ in the (Gln) form, because treatment with the solvents used to extract neurotensin from hypothalamic tissue led to the conversion of the (Gln) peptide

Although it is a primary concern when making biologically active peptides, racemization has not generally been observed during solid phase synthesis (22). However, asparagine and /3-benzyl aspartic acid in synthetic peptides have (23-24) been known to undergo a-8 rearrangement during the HBr cleavage reaction. We have good evidence that neither of these undesira- ble side reactions occurred to any appreciable extent during this synthesis, because both major products possess intact asparagine residues and were completely degraded to free amino acids by enzymes specific for the L configuration and LY linkage.

The availability of synthetic material has now made possible the development of a radioimmunoassay for neurotensin (25) and should greatly facilitate the many studies that must be done to pursue the physiology and biochemistry of this interesting peptide.

Acknowledgments-We are grateful for the excellent techni- cal assistance afforded by Ms. Linda Gillespie and by Mr. James Goldman. For generous instruction and informative discussion concerning methods of peptide synthesis as well as a gift of chloromethylated resin we owe a special thanks to Dr. Arnold Marglin. We also thank Dr. Robert Fellows for his gift of purified pyrrolidonecarboxylyl peptidase.

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R Carraway and S E LeemanThe synthesis of neurotensin.

1975, 250:1912-1918.J. Biol. Chem. 

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