D A V I D H. C O Y , 1 E S T H E R J. C O Y , K A t

Department o f Medicine, Tulane University, and *The Isaac Gordon Center for i

University of Rochester School o

R e c e i v e d 3

coY, D. H., E. COY, K.-Y. LEE AND W. Y. CHEY. Soli~ hormones: Secretin and motilin. PEPTIDES 3(2) 137-141, 191 in the region of 20-40 amino acid residues have now been methodologies but, particularly, new and powerful purifical preparative routes for the numerous neuro-gastrointestinal obtained in 16% and 10% yields, respectively, after simplifieq by gel filtration followed by preparative high performance liq by TLC and analytical high performance liquid chromatogra tested against a standard sample of natural material for effe biological activity on interdigestive motility in the dog. Secn bioactivity during purification and prolonged storage. We ob tion schedule and have found no loss of purity of peptide w

routinely reporteO. Utlllzmg sta ~urification techniques we have been de

peptides. In the present study, ~lified two-step purification of hydrog~

uid chromatography. Peptides we phy. Secretin was found to have

effects on pancreatic secretion in the Secretin has been reported to undergo

We observed no obvious problems duri ~tide which has been kept for 6 months

Solid-phase synthesis Biological activities

~- and 22-residue peptides with size range of secretin, pat ~own in Fig. 1. Historically, sequence [6], we decided t hormonal agents with demon- secretin. In an effort to vith other gastrointestinal pep- problems during long purif properties [13], a number of purification schedule was I

ae structurally related vasoac- reverse phase chromatogr ~nd glucagon. The primary ef- bonded silica, developed l on release of bicarbonate and purification of somatostatir ine pancreas and inhibition of Motilin was characteriz~ lease. Clinical trials on the use a more limited and useful th bleeding ulcers have shown tions than secretin. PrinciI unpleasant side-effects have gastrointestinal motility [5]

.~rapeutic investigations on the synthesis of motilin has b perhaps been severely limited 1.8% yield was obtained, l a shortage of genuinely pure, evaluation of our preparati

by synthesizing it in simil~ of secretin have been reported ation approaches or repetitive [4]. One previous solid-phase .~d [9]; however, possibly some Solid-phase syntheses lop rapid solid-phase methods model 990 automatic pepti conflicting reports on the in- was performed on a Wat

Tltilizint~ e.xnarle.nc.a oa inad in c h r n m a t o a r a t ~ h e o u i n n e d

dilute acetic acid.

Secretin Motilin

SECRETIN and motilin are 27- the amino acid sequences shown secretin is the oldest of the hormon~ strable biological effects. As with othe tides, it has many biological which overlap with those of the tive intestinal peptide (VIP) and fects of secretin appear to be protein releas-efrom the exocrlne gastrin-induced gastric acid release. of secretin to treat patients with some promise [1]; however, been noted [15]. Extensive thera peptide and its analogs have perhal: by difficulties in synthesis and natural material.

Several classical syntheses of secre using either fragment condensation coupling of single residues [14 synthesis has also been reported reluctance to intensively develo for secretin has arisen due to stability of the peptide [2,8]. Utilizing the solid-phase synthesis of a large nut

l~end re.nrint reauest~ to Dr. David H. C

n the U.S.A.

ase Synthesis of Gastrointe Secretin and 1

K A E - Y O L I_

School q Gastroen

of MediciJ

M a r c h 1

Solid-phase syt 1982.--Many

routinel,

onal ~ther

Biological Hormone

.in

W I L L I A M Y. C H E Y *

New Orleans, LA 7011~ ;enesee Hospital !er, N Y 14007

~logical activities of gastroinle~ id-phase syntheses of peptide c zi~ standard solid-phase synt

developing rapid and effi ty, secretin and motilin

lydrogen fluoride-cleaved Pel~ ptides were essentially homoge~

full biological activity ' the dog. Motilin exhibiteq • go rearrangement with 1¢

:ms during our abbreviated par as powder lyophilized

~articularly the closely to carry out a similaz circumvent potential

}urification routes, a shc used which employed

graphy on octadecyls from earlier method

ttostatin-28 and catfish soma acterized much more recent

range of known bi¢ ipal among them are

! [5]. Since only one oth~ been reported [10] in

we also undertool ~arative and purification rr similar fashion to secretin

METHOD

were carried out oi ~tide synthesizer. Ana

Waters Associates mod ipped with two 6000A pump miner. Preparative HPLC w

:hooi of Medicine, New Orleal

Motilin

1. Amino acid sequences of secretin and motilin.

kltex high pressure glass columns pumped with a Fluid ~ring Co. pump and pulse yses were run on a Beckman model 119CL analyser and alated with a System AA computing integrator.

ide Synthesis and Purification

ecretin, which is an amide (Fig. 1), was assembled wise on 1.0 mmole of 1% cross-linked benzhydrylamine I (Bachem) using a schedule of events described [6] for VIP synthesis, except that two treatments (for 1 and 25

33% trifluoroacetic acid in methylene ride were used during the a-amino deprotection step. no acids were coupled as their Boc derivatives (Bachem) functional side-chains were protected as follows: Arg His, tosyl; Asp and Glu, benzyl; Ser and Thr, benzyl. a amino acid was successively coupled in the presence of }propylcarbodiimide and, in the case of Box-Asn or Gin, equivalent of 1-hydroxybenzotriazole. Coupling reac-

. . . . o . . . . . . . . . . . step by using the ninhydrin test ncomplete within 1 hour were ~priate symmetrical anhydride ure in DMF [6]. Although few ~locking was necessary, free 0.25- er the above processes were tcetylimidazole. solid-phase synthesis, a 0.25 fin was treated with hydrogen

anisole for 30 min at 0 °. After stream of nitrogen, free, de-

s precipitated by addition of into 5 ml of 2 M acetic acid.

Lo a column (2.5×95 cm) of ,as eluted with 2 M acetic acid. ae void volume as a major peak contained a major component

the solvent system • id:water (15:10:3:12). This column (2.5x45 cm) of ODS

!4/~m) which was eluted with a 350 ml each of 15 and 35%

m acetate (pH 4) at a flow rate e of about 60 psi. Eluate was o • Fractions were examined by

1 ¢ ~ U l

our V min each) with chloride Aminq and and Each; diiso one tions were monitored at each ste [11]. Couplings which were incom recoupled by using the appropriate pre-formed at room temperature instances occurred where blocki~ amino groups remaining after terminated by reaction with ac,

After completion of the mmole portion of peptide-resm fluoride (30 ml) containing 10% anisok rapid removal of H F under a protected peptide amide was ether, washed and extracted This solution was applied to Sephadex G-25 (fine) which was elute Material emerging just after the void (254 nm) weighed 457 mg and by TLC on silica using l-butanol:pyridine:acetic acid:wateJ material was injected onto a silica LRP-1 (Whatman) (13-24 linear gradient formed from 1-propanol in 0.1 M ammonium acetal of 5 ml per rain and pressure monitored at 254 nm (Fig. 2). TLC and analytical HPLC and the r emerging at 375 ml clearly contained which was identical to a standard san

Lyophilized peptide from this peak

lu-Leu-Ser-Arg-Leu-Arg-

eu-Gln-Gly-Leu-Va I-NH2 O.2S-

lu-Leu-Gln-Arg-Met-Gln-Glu-

o ~

o 20o

FIG. 2 dampener. Amino acid umn (2

dient oJ ml per

0

E c

g t ' q

o

sole

uted with 2

~o ,;0 v o u J I / ~

tm of crude synthetic secretin, Whatman LRP-10DS silica w panol in 0.1 M ammonium aceU

!

7 5 ~

VOLUME (ml)

m o f crude synthet ic mot i l in e|u man LRP- ] ODS sil ica wi th a li I 10% acetic acid. Flow rate; 2

0 5 -

E

| ! I ! i

12 24 0 12 24

4. Analytical HPLC of purified secretin (40/.~g) and motilin (25

present.

Bioassays

Secretin was tested in prepared with gastric and scribed previously [1]. At thetic secretin or Kabi's p~ ng/kg was administered ir in a hind leg. While the ga the experiment, pancreati creatic fistula in 15 min sa

ff LiChrosorb ODS silica (10/~m) of bicarbonate was deter ~er 20-35% and 15-30% 2-propanol, and bicarbonate output 1 x ic acid. Flow rate: 1.5 ml per rain. Synthetic motilin was a

prepared with gastric cm implanted on the anterio] [12]. A myoelectric recor terdigestive myoelectric c

ed on the original incorporation ranged from 70 min to 9 • Analytical HPLC on a column second phase III [12], syr

RP-18 dose of 0.06/~g/kg/hr [12] ~tic acid system gave a sharp samples were drawn for r oximately 1% by area of a later lin concentrations [12]. his could be readily removed by )arative column. sample of peptide hydroloyzed RESULTS g acceptable amino acid ratios: ;er, 3.15(4); Glu, 2.87(3); Gly, Our results indicate tt 4(1); Leu, 6.05(6); Phe, 0.85(1); two more members of th~ mino acid analysis of a leucine tides that can be readily Asp, 1.93; Glu, 1.05; Gly, 2.00; solid-phase methods folio .11; Phe, 1.01; His, 0.92; Arg, with HF, a reagent whicl

due to its harshness and te lized peptide 6 months after its This standard methodolo~ ad TLC revealed no alterations in cost, however, compan

in the solid-phase peptide

TIME (rain)

FIG. 4. t~g) on a column (0.4×25 cm) of eluted with linear gradients of either respectively, in 0.1% trifluoracetic

represents a yield of 16% based of the benzhydrylamine resin. Anal (0.4x25 cm of LiChrosorb 2-propanol-0.1% trifiuoroacetic peak contaminated with approximate eluting component (Fig. 4). This coulc recycling peptide on the prep

Amino acid analysis of a in 6 M HC1 gave the followin Asp, 2.17(2); Thr, 1.65(2); Ser, 1.92(2); Ala, 1.00(1); Val, 0.94(1 His, 0.88(1); Arg, 3.91(4). Amino aminopeptidase digest gave: Asp, Ala, 0.98; Val, 0.95; Leu, 6.1 3.98.

Examination of the lyophilized pe final purification by HPLC and in properties or purity.

Motilin was assembled on a benz port in a similar fashion with Boc-a-b the first amino acid to be incorpoi C-terminal Gin could be directly fort ~ 6 1 . . ^ ~--^1 a _ _ ~ . ' ~ l _ c _ A _ . i . . . . . . . _

~ITHESIS, ACTIVITY

Aft from q partial was p~ with a 1-prop Exami major tions v motili]

Thi similal be ess 3 solw with tl lowed (4:1:1) (I:1:1:

Art gave: Gly, i 0.96(1 i n 4 M dues,

(10 txm) using a

ateb mid

f the final Boc group, motili of resin by standard HF I an Sephadex G-25. Furth¢ a LRP-1 column (1.5x45 c nt formed from 200 ml each acetic acid at a flow rate of fractions by TLC and HPI

in the third peak (280 nm) ( or maximum purity to give

as examined by analytical :o those used for secretin an ageneous (Fig. 4). TLC was )n silica gel plates• One spot Rt's after visualization with ch reagent: 1-butanol:acet ~utanol:acetic acid:water:l ropanol:0.1 M acetic acid (~ Llysis of a sample hydrolyze ; Thr, 1.00(1); Glu, 6.06(6) 0.73(1); Met, 0.91(1); Ile,

2); Lys, 1.86(2); Arg, 1.93(~ fonic acid gave a similar val ~t, indicating that little Met0

an 18 hour-fasted, ( chronic pancreatic :

After a 30 min contro rare natural secretin i intravenously via a c~ astric cannula was le:

mcreatic juice was collecte, samples. Volume and

determined as reported 1 ~r 15 min was calcul assayed in 3 fasting, c

:annulae and monopo anterior wall of the proxin

recording was made of oi complex. The duratio 90 min and at 20--25 'nthetic motilin was in

12]. In two dogs, serial radioimmunoassay oJ

AND DISCUSSION

:hat secretin and tool of the growing series o f ]

ty prepared by relati followed by cleavage an(

which has often been cri and tendency to degrade s

,gy results in a consic 'ed with more recent

atide field involving new derivatives, as long as reae he 16% yield of secretin obt ement over the 10% yield ,er [9] in their previously r d possibly reflects our abbr

4 0 0 -

.300

200

.I00.

0 , ~ . . . . o , 23 5;7 ;,o

15 MINUTE INTERVALS

5. Pancreatic secretion of bicarbonate in a dog wi th chronic reatic fistula in response to synthetic secretin and Kabi's nat- secretin in a dose of 0.03 U (7.5 ng)/kg. Mean of two experi-

,n nrocedure involvinlz fewer stens and losses. The nres-

nm, as in analytical HPL( and off-scale peaks. The fleet true peptide contents components absorb much reactions with aromatic b actions during H F treatm~

As shown in Fig. 5, the ate in response to IV injec comparable or even great~ dose of a natural secretin

~er step and 1o 1~ ous instability of secretin b much higher than the 2% yield tion of secretin in solution ~rted by Ikata et al. [10]; how- erally been attributed to a )rimarily used to evaluate a boxyl group on Asp in p( tsing new phosphorus coupling reaction can be separated

we could find no evidence ?LC at intermediate pressures purification procedure wa', ates the purification of a crude al. [2] have demonstrated the most critical stage in the some cleavage of the Asp

prides prepared by solid-phase pH. They suggest that a m t in complex mixtures of con- biological activity which | gradients of the organic eluting tion of peptide to glass or ( :able to most peptides thus far solutions used for bioass~ lifications. Generally, we find nomenon with many pepti ms applicable to a wide variety The IV infusion of out 0-20% acetic acid or 0.1% tri- expected induction of pha ts component with the choice of the duodenum (Fig. 6). T! die as the organic component, tivity found in previous s ,olatile. LRP-1 columns can be prepared by solution metL 1ours and elution profiles, such 4, obtained with loads of sev-

lly be followed at the standard This research was support aromatic residues present in a would like to thank Dr. Rolf (

aent of absorption at 215-220 of classically synthesized sec

R i~,b-T, R i~,N C F,~

FIG. pancreatic ural ments.

cation procedure tg fewer ent 10% yield of motilin is also much h of the peptide which was reported ever, their synthesis was primaril> fragment-coupling approach usm reagents.

The use of preparative HPLC greatly simplifies and accelerates peptide. This is undoubtedly synthetic process of large peptides since they are always present taminants. The same general g component are ususally applicable to encountered with minor modification that the two best elution systems of peptides comprise either 10-20 fluoroacetic acid in the aqueous corn either 1-propanol or acetonitrile These media are completely volatile. run within the space of 2 or 3 hours an as those shown in Figs. 2 and 4. eral hundred mg, must normaU absorption wavelengths of the particular peptide. Measurement

1. Becker, H. D. Secretin in the treatmev rhage. In: Cimetidine, edited by W. C Amsterdam: Excerpta Medica, 1978,

KABI SECRETIN (.03 U/kg) >.

I-- I O O % -

i 50%- ~u Q.

0

~ 50%.

Q.

FIG. 6 infusiol motilin compal state.

|

II 12

h hi

rily

tions.

IV. MOTILJN [ O ( ~ j

I i MOTIL

I I 60 90 120

TIME IN MINUTES

phase l I I - l ike act ivi ty of the dl moti l in in a dose of 0.06/~g/kg/] plasma immunoreactive motilin yclic increase of motilin in th,

HPLC, results in too grea absorption profiles tl

of the peaks obserw more strongly due p

blocking groups and atment.

the pancreatic secretio~ ection of our synthetk

reater than that produce, preparation. The sul in solution and duri and during lyophiliz

a-/3 rearrangement thl ,osition 3. The by-pr~

by reverse-phase E rice of it occurring duri] was completed. Indeed,

that very little ream p-Gly bond only take: more likely explanatio

have been reported 1 other materials when

bioassay. This is a not unq ~tides.

our synthetic motilin r~ 3hase III-like myoelect This was indistinguish

studies [5] using syn methods.

A C K N O W L E D G E M E N T S

)ported in part by NIH grant Rolf Geiger for helpful discu.'

secretin.

'.., P. Kranenburg and D. Vo tin, a gastrointestinal hormon ~. Brunfeldt. Copenhagen: Scr

I C I ~ , l i p . tY4D"~JO, IMOU.

,'oy, D. H. and J. Gardner. Solid-phase synthesis of porcine ,asoactive intestinal peptide. Int. J. Peptide Protein Res. 15: '3-78, 1980. -'oy, D. H., W. A. Murphy, C. A. Meyers and J. L. Fries. ;ynthesis and some biological properties of somatostatin-28 and ',atfish somatostatin I. In: Peptides 1980, edited by K. Brun- eldt. Copenhagen: Scriptor, 1981, pp. 308-314. }rossman, M. I. Stability of secretin. Gastroenterology 57: 767, 969. temmasi, B. and E. Bayer. The solid phase synthesis of por- ',ine secretin with full biological activity. Int. J. Peptide Protein ?es. 9: 63-70, 1977.

4THESIS, ACTIVITY

Dryburgh. Motilin, a gastric 10, Ik the complete amino acid se- an

-537, 1973, Sy e and T. M. Chang. Effect of dip andial pancreatic secretion in Ch 275, 1979. 11. Ka ilin. In: Clinics in Gastroen- Co ltzfeld. London: W. B. Saun- sy~

12. Le mu shi tivi 19;

13. Mt tro

Ra 14. Va

tid Wi

15. W~ s e ¢

eg~ lro

)iri, S. Yamada and S, Tachibat gXI, Phosphorus in organic s' 'cine m0tilin by the solid.phas lorazidate and diethyl phosr ull. 28: 3347-3356, 1980. ,. Colescott, C. D. Bosinger a Lection of free amino groups in ides. Analyt. Biochem. 34:595 Y. Chey, H. H. Tai and H. Y~ atillin: Validation and studies q sma motilin and interdigestive denum in dog, Am. J. dig. Di

a: isolation, structure, and fum mones, edited by G. B. J. GI~ ;0, pp. 85--126. J H. C. Beyerman. REMA sy es 1974, edited by Y. Wolm~ 1975, pp. 165-172.

sad M. Rothmund. Effect von ten blutungen aus magen un prospektiven altemierenden s 41, 1980.