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Page 1

Handbuch der experimentellen Pharmakologie
Handbook of Experimental Pharmacology

Heffter Heuhner New Series


Herausgeber Editorial Board

O. Eichler, Heidelberg' A. Farah, Rensselaer, NY
H. Herken, Berlin· A. D. Welch, Princeton, NJ

Beirat Advisory Board

E. J. Ariens . Z. M. Bacq . P. Calabresi . S. Ebashi . E. G."Erdos
V. Erspamer . U. S. von Euler' W. Feldberg

G. B. Koelle' O. Krayer . T. A. Loomis' H. Raskova . M. Rocha e Silva

F. Sakai' J. R. Vane' P. Waser . W. Wilbrandt

Page 2

Secretin, Cholecystokinin,
Pancreozymin and Gastrin


M. Bodanszky, R. Carratu, D. A. Dreiling, R. Fussganger,

H. D. Janowitz, J. D. Jamieson, J. E. Jorpes, V. Mutt, E. F. Pfeiffer,

J. Plessier, M. L. Ramorino, S. Raptis, A. Torsoli,
M. Zimmerman

J. E. Jorpes and V. Mutt

With 135 Figures

Springer-Verlag Berlin. Heidelberg. New York 1973

Page 192

Synthesis of Secretin by the Stepwise Approach 183

Many biologically important peptides (oxytocin, vasopressin, vasotocin,
gastrin, secretin, physalaemin, eledoisin, etc.) have an amino acid amide as their
C-terminus: therefore, this problem cannot be belittled. In an early model experi-
ment (BODANSZKY and SHEEHAN. 1964), the protected C-terminal tripeptide
sequence of oxytocin, benzyloxycarbonyl-L-prolyl-L-Ieucylglycine was built up
on the solid support and the peptide was separated from the resin by treatment
with a methanolic solution of ammonia. The product, the protected tripeptide
amide, was obtained in moderate yield, but the experiment was still somewhat
encouraging. On the other hand, while this tripeptide had glycine as its C-terminal
amino acid, in secretin valine is in the same position and it is well known that
steric hindrance by the isopropyl side chain of valine is quite noticeable in the
aminolysis and ammonolysis of valine esters. Therefore first the ammonolysis of
benzyloxycarbonyl-L-valine-resin ester was examined. In about a day all the
protected amino acid was removed from the resin, but only about one fifth of
the product turned out to be the desired amide, the major portion was found to
be benzyloxycarbonyl-L-valine methyl ester. Seemingly, the steric hindrance
prevails more in ammonolysis than in trans esterification. Of course this gave no
serious reason for concern since by prolonged ammonolysis the methyl ester could
be converted into the desired amide. In the next experiment the protected C-
terminal hexapeptide sequence of secretin, L-Ieucyl-L-Ieucyl-L-glutaminylglycyl-
L-Ieucyl-L-valine, was built up on the MERRIFIELD resin. In the synthesis of this
sequence p-nitrophenyl esters of the protected amino acids were applied for
acylation and the completion of each coupling was ascertained by the determin-
ation (UV absorption) of p-nitrophenol in the filtrate. The unreacted excess of
the active ester was determined similarly. The protected hexapeptide on the resin
was treated with methanolic ammonia. This time all the material was isolated
as methyl ester, and practically no amide was found. The amide, however, could
be made by a prolonged exposure of the protected hexapeptide methyl ester to
ammonia in methanol. Obviously the combined hindering effects from the valine
side chain, the resin matrix and the peptide chain led to this result (BODANSZKY
and SHEEHAN, 1966).

Since it still seemed to be possible to continue the synthesis in this manner,
the stepwise lengthening of the peptide was continued until the tridecapeptide
sequence, tert-butyloxycarbonyl-~-benzyl-L-aspartyl-O-benzyl-L-seryl-L-alanyl-
nitro -L-arginy 1-L-leucyl-L-glutaminyl-nitro -L-arginyl-L-Ieucyl-L-Ieucyl-L-gluta-
minylglycyl-L-Ieucyl-L-valine attached to the resin by an ester bond was prepared.
In this case the chain withstood many vigorous attempts and could not be removed
by ammonolysis or ester exchange. That the peptide was indeed present on the
polymeric support was shown by its removal with hydrobromic acid in acetic acid,
but this treatment resulted of course in a peptide acid and not in the peptide
amide which is necessary for the synthesis of secretin. At this point our attempt
to synthesize secretin on a solid support had to be abandoned.

D. Synthesis of Secretin by the Stepwise Approach
(Through Isolated Intermediates)

In most respects this synthesis (BODANSZKY and WILLIAMS, 1967; BODANSZKY,
ONDETTI, LEVINE and WILLIAMS, 1967) was based on the synthesis of oxytocin
by the stepwise nitrophenyl ester method (BODANSZKY and DU VIGNEAUD, 1959).
The first amide bond was produced by the ammonolysis of benzyloxycarbonyl-L-

Page 193

184 M. BOD.ANSZKY: The Synthesis of (Porcine) Secretin

valine p-nitrophenyl ester. The protecting group was removed from the resulting
amide by treatment with hydrobromic acid in glacial acetic acid and the next
amino acid L-Ieucine was introduced via benzyloxycarbonyl-L-Ieucine p-nitro-
phenyl ester. The chain was lengthened in the same manner until the C-terminal
hexapeptide amide, L-Ieucyl-L-leucyl-L-glutaminyl-glycyl-L-leucyl-L-valinamide
was in hand. All the intermediates so far were secured in excellent yield and in
crystalline form. The next amino acid was applied as benzyloxycarbonyl-L-
arginine 2,4-dinitrophenyl ester (BODANSZKY and ONDETTI, 1966). The nitro-
guanidine derivative was chosen as the protected form of arginine because the
nitro group can be removed by hydrogenolysis, a rather mild operation. The
sensitivity of secretin to sodium in liquid ammonia was unknown and therefore
the tosyl-protection of arginine could not be considered. The different side chain
protecting groups were selected in such a way that all should be removed in a
single step by hydrogenolysis at the completion of the synthetic procedure. These
considerations, the nitro protecting group on the arginine moieties introduced
some limitation in the choice of methods of activation: the p-nitrophenyl ester
of benzyloxycarbonyl-(and of t-butyloxycarbonyl)-nitro-L-arginine is difficult to
prepare and is unstable (BODANSZKY and SHEEHAN, 1960). The 2,4-dinitrophenyl
esters are more suitable intermediates; they still show some shortcomings, e. g.
they are readily hydrolysed by traces of water that is often present in the comm-
only used solvent, dimethylformamide. The L-threonine residues were also in-
corporated through 2,4-dinitrophenyl esters. The hydroxyl group of L-serine, the
side chain carboxyl groups of the glutamic acid and aspartic acid residues were
all protected by benzyl groups.

The protected hendecapeptide derivative benzyloxycarbonyl-L-alanyl-nitro-
glycyl-L-Ieucyl-L-valinamide was the last intermediate that was prepared with
benzyloxycarbonyl (BERGMANN and ZERVAS, 1932) as aminoprotecting group.
Since the nitro group on the arginine residues had to be kept intact all the way
during the chain lengthening procedure, hydrogenolysis could not be applied for
the removal of the benzyloxycarbonyl protection. The only remaining practical
method, treatment with hydrobromic acid in acetic acid leads to a partial 0-
acetylation of the serine residues. Therefore when in the preparation of the pro-
tected dodecapeptide a derivate of L-serine had to be chosen, O-benzyl-L-serine
was protected on its amino function not by the benzyloxycarbonyl but by tert.
butyloxycarbonyl grouping (MoKAy and ALBERTSON, 1957; ANDERSON and
MOGREGOR, 1957; SOHWYZER, SIEBER and KAPPELER, 1959), because the latter
can be removed by a comparatively mild treatment with acids, e.g. with trifluoro-
acetic acid at room temperature within a few minutes. Of course the tert. butyl-
oxycarbonyl protection had to be applied throughout the rest of the-lengthening
of the chain, except in the addition of the N-terminal amino acid, histidine. For
the preparation of the last protected intermediate, the heptacosapeptide derivative
N- benzyloxycarbonyl-L -histidyl-O- benzyl-L- seryl-~-benzyl-L-aspartyl-glycyl-L-
threonyl- L- phenylalanyl- L- threonyl- 0- benzyl- L-seryl-y-benzyl- L-glutamyl- L-
leucyl- 0 -benzyl- L- seryl- nitro -L-arginyl- L-Ieucyl-nitro -L- arginyl-~ -benzyl- L-
aspartyl- 0 -benzyl- L-seryl- L-alanyl-nitro- L-arginyl- L-Ieucyl- L-glutaminyl-nitro-
L-arginyl-L-Ieucyl-L-Ieucyl-L-glutaminyl-glycyl-L-Ieucyl-L-valinamide the ben-
zyloxycarbonyl group needs no selective removal. It is lost during the hydro-
genolysis used for the removal of the benzyl protecting groups from the amino
acid side chains. Therefore the histidine residue could be attached to the partially
protected hexacosapeptide as benzyloxycarbonyl-L-histidine azide (HOLLEY and
SONDHEIMER, 1954), or as bisbenzyloxycarbonyl-L-histidine p-nitTophenyl ester.

Page 383

Subject Index 375

Pancreatic, secretory proteins, passage of
the zymogen granules identical to those
of pancreatic juice 196

-, zymogen granule discharge 209-214
-, -, metabolic requirements 214-216
Pancreatic fistulas 2, 41
Pancreatic juice
-, calcium secretion 64, 220
-, cytological examination 232,233
-, coagulability by heat 1
-, composition 64-66,67-74,220
-, digestion of starch 1, 62
-, digestion of fats 1, 2, 62
Pancreatic secretion after vagotomy 4, 40
-, stimulation by acetic acid in duodenum 1

, caerulein 82-83
-, - gastrin 80-83
-, - hydrochloric acid in duodenum 2
-, - pentagastrin 81-82
Pancreatography with intraceliac injection of

CCK 331
- with intraceliac injection of secretin 331
Pancreotest Astra 218, 226, 229
"Pancreozymin" 25,27, 75, 80, 219, 234,

-, dose response curve 237
-, preparations Boots 219,238
-, - GIH Res. U (CCK-PZ) 219
Pentagastrin, action on the pancreas 81-

-, effect on the gastric acid secretion 121
Pentapeptide IeI 50, 123, 59-60
-, action on the gallbladder 95
-, effect on gastric acid secretion 107
Pepsin secretion, effect of CCK 109
-, - gastrin 109
-, - glucagon 112
-, - secretin 110-112
Perfusion of pancreatic duct 69
Pernocton® 64
Phyllocaerulein 30
Phyllokinin 30
Phyllomedusa sawagi 30
Physalaemin 30, 183
Pilocarpin 55
Pituitrine, discovery 4
Plasma insulin level in insulinoma patients

increases after caerulein 279
Polyethylene glucol (PEG) in the secretin

test 230
Potentiation of effects of gastrointestinal

hormones 119-123
-, the effect of gastrin and CCK on gastric

secretion by cholinergic drugs 122
-, - histamine on gastric acid secretion

-, - CCK on gallbladder by secretin

-, - secretin on pancreas by CCK 122-

-, vagal stimulation 120-121
-, the vagal effect by gastrin 119-120
Probanthine in cholangiography 327,328
Procaine, relaxation of sphincter Oddi by


Prose cretin 44
Prostigmine in cholangiography 328
Pyruvate 72

Raja clavata 34
Rehfuss tube 221
Renin, discovery 4
Royer camera for transvesicular cholangio-

graphy 330

Scintigraphy of the gallbladder, CCK in

- of the pancreas, CCK in 334, 335
SOP, synthetic octapeptide of "pancreo-

zymin" 282
Secretin, action on the bile flow 86-92,223
-, - Brunner's glands 85-86
-, - gallbladder 96,223
-, - ion flux through the intestinal

epithelium 85
-, - insulin release 131, 132, 262
-, absorption from intestine 49
-, adsorption on alginic acid 10
-, amino acid composition 12
-, attempts at purification 6-9
-, bioassay in dogs 133-134
-, - in cats 134-135
-, - in rats 135-137
-, breakdown and synthetic products 12,

-, chemical structure 12, 14, 45, 180
-, -, proposed conformation 192
-, -, structural conformation 15,190-

-, -, structural similarities glucagon-

secretin 28, 31, 192
-, choleretic effect 16-17,266
-, content in hypophysectomized rats 35
-, discovery 3-6
-, distribution in the intestine 35, 36
-, effect on gastric acid secretion in the dog

, in man 101-103

-, - in the cat 103
-, - in the rat 103-104
-, on FFA, free fatty acids, in plasma 284
- on serum insulin in normal subjects 284
- - in onset diabetes 284
-, half life time 49, 50, 267
-, hydrogen peroxide, action on 26
-, hypoglucemic action of 7,8
- in birds 35
- in mucoviscidosis 34
-, isolation 9-12
-, lipolytic activity 30, 267
-, molecular weight 14, 218, 266
-, occurrence 34, 35
-, preparations
-, -, Astra (Pancreotest) 218, 226, 229
-, -, Boots 219, 229, 230, 232
-, -, Byla 218
-, -, Eli Lilly 218, 227, 229, 231
-, -, GIH Res. U. 65, 138, 218, 219, 227,

231, 232, 238
-, -, Vitrum 139, 219, 229, 230

Page 384

376 Subject Index

Secretin, preparations Wyeth 9, 218, 226
-, purification by means of electrophoresis

-, - CMC 10, 11
-, -, countercurrent distribution 9, 10, 11
-, -, DEAE Sephadex 11
-, release of 41-42,44-49
-, route of administration 231-232
-, standards 137-138
-, synthesis of 15, 181-194
-, -, attempted synthesis on a solid

support 183
-, -, the stepwise synthesis of porcine

secretin 183-187
-, -, synthesis by fragment condensation

-, synthetic secretin with fJ-aspartyl instead

of a-aspartyl 30
secretin test for pancreatic function,

methodology 221-223
-, submaximal secretin test 223-228
-, maximal secretory response 228
-, in inflammatory and neoplastic disease

-, in chronic pancreatitis 233
-, in carcinoma of the pancreas 233, 234
-, secretin-pancreozymin test 236-238
-, pancreozymin-secretin test 238-240
-, serum-enzyme test 240,241
-, tryptic and thrombinic peptides of

-, units -, -, the clinical unit of Lagerli:if

8, 138-140,225
-, -, the Crick, Harper and Raper unit

-, -, the Hammarsten cat unit 8, 9, 134,

-, -, the Ivy cat unit 9, 134
-, -, the Ivy dog unit 9, 133-134
-, -, the rat unit of Debray et al. 136
-, -, - of Love and Heatley 135-136
-, -, comparison between units 138-140

Secretory proteins of the exocrine pancreatic
cell, transport of and discharge 195-

Sphincter of Lutkens 312, 325
Sphincter of Oddi, action of CCK on 28,

Sphincterotomy 327, 328
S-cells, producing secretin 39
Streptozotocin diabetic rats 295
Subcellular fractions carrying secretin or

gastrin 39
- organelles in the exocrine pancreatic cell


Theophylline 74,75
Thiry Vella duodenal loop 40,41
Thoracic duct, nonparticipitation in the

uptake of secretin and CCK 50, 51
-, lymph flow, effect of secretin and CCK on

TOT, intestinal factor increasing glucose

induced insulin release 293
Toxic effects of crude secretin and CCK

preparations 133
Trypsinogen 44, 62
Tyrosin ester sulfate 25, 29, 33, 34

Urecholine 60, 80, 121
Urocholecystokinin 24, 144, 323

Vasopressin, action on pancreatic secretion 84
-, synthesis 182
Vasotocin 183
Vitrum Secretin 139

Wyeth Secretin 9, 218, 226

Xenopus laevis 29
X-ray analysis of the small intestine 131-

Xylocaine in cholangiography 328

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