Natural Product
`Chemistry
`
`Proceedings
`of the First International Symposium
`and Pakistan-US. Binational Workshop,
`Karachi, Pakistan
`
`Edited by
`Atta—ur-Rahman
`
`Springer-Verlag
`Berlin Heidelberg NewYork
`London Paris Tokyo
`
`'
`
`MA1A Exhibit 1023
`MAIA V.BRACCO
`IPR PETITION
`
`
`
`
`MAIA Exhibit 1023
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`Professor ATTA-UR—RAHMAN
`
`H.E.J. Research Institute of Chemistry
`University of Karachi
`Karachi-32, Pakistan
`
`With 142 Figures and Numerous Schemes
`
`ISBN-13: 978-3-642-71427-6
`DOI: 10.1007/978-3-642-71425-2
`
`e-ISBN-13: 978-3-642-71425-2
`
`Library of Congress Cataloging-in-Publication Data. International Symposium and Paki—
`stan-US. Binational Workshop on Natural Product Chemistry (1st : 1984 : Karachi, Paki-
`stan). Natural product chemistry. “The first International Symposium and Pakistan-US.
`Binational Workshop on Natural Product Chemistry
`February 1984”—Pref. Includes
`index.
`1. Natural products—Congresses.
`I. Rahman, Atta-ur-, 1942-
`.
`11. Title.
`QD415.A1147
`1984
`547.7
`86-20420.
`
`This work is subject to copyright. All rights are reserved, whether the whole or part of the
`material is concerned, specifically those of translation, reprinting, re-use of illustrations,
`broadcasting, reproduction by photocopying machine or similar means, and storage in data
`banks. Under § 54 of the German Copyright Law, where copies are made for other than
`private use, a fee is payable to “Verwertungsgesellschaft Wort”, Munich.
`
`© Springer-Verlag Berlin Heidelberg 1986
`Softcover reprint of the hardcover lst edition 1986.
`
`The use of registered names trademarks, etc. in this publication does not imply, even in the
`absence of a specific statement, that such names are exempt from the relevant protective
`laws and regulations and therefore free for general use.
`
`Offsetprinting: Druckhaus Beltz, Hemsbach/Bergstr.
`
`2131/3130—543210
`
`
`
`
`

`

`Atta-ur-Rahman (ed.), Natural Product Chemistry
`© Springer-Verlag Berlin Heidelberg 1986
`
`-255-
`
`
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`

`

`256
`
`The main structural characteristics of the gastrin—cholecysto—
`
`kinin family of peptide hormones are:
`
`1)
`
`the common C—terminal
`
`tetrapeptide amide sequence Trp—Met—Asp—Phe-NH2 which already
`
`in early structure—function studies on gastrin was recognized
`
`as the minimum fragment with physiological activity although
`
`at low potency, and thus as a kind of active center of the hor—
`
`mone (1);
`
`2)
`
`the tyrosine—O—sulfate residue which for chole-
`
`cystokinin (CCK) represents an essential structural feature for
`
`full hormonal expression, whereas for the gastrins its role has
`
`not yet been identified; they are isolated in the nonsulfated
`
`form I as well as in the sulfated form II (2,3) and no signifi—
`
`cant differences in their physiological actions have been de—
`
`tected so far;
`
`3)
`
`the complex size-heterogeneity as resulting
`
`from the posttranslation processing of their precursor mole-
`
`sequence
`
`shades! active
`
`Fig. 1. Primary structures of A) human gastrins (HG) and B)
`
`porcine cholecystokinins. Arrows indicate enzymatic
`
`cleavages of the prohormonal molecules with production
`
`of gastrins and cholecystokinins of decreasing Chain—
`
`length. The C-terminal amides result from enzymatic
`
`processing of glycine-extended forms
`
`-256-
`
`-256-
`
`
`
`
`

`

`257
`
`cules (Fig. 1)
`
`(4). To better understand the structural bases
`
`for the biological actions of this class of hormones and to pos—
`
`sibly identify the physiological significance of the observed
`
`heterogeneity both in respect to sulfation and extension at the
`
`N—termini, extensive synthetic studies have been performed in
`
`the last years in our laboratory (5 -15). Aim of these inten—
`
`sive efforts was
`
`i)
`
`to obtain the various components of this
`
`class of hormones at the highest possible degree of purity for
`
`structure-function studies
`
`ii) to identify fully active ana—
`
`logs stable on storage and handling as needed for physiological
`
`and clinical studies, and finally iii) to synthetize deriva-
`
`tives well suited for an efficient improvement of the immuno—
`
`logical methods necessary for a reliable quantitative and selec-
`
`tive evaluation of the different components both when circula-
`
`ting in the plasma and in the tissue of their biosynthesis. Our
`
`contributions in the field are reviewed in the following:
`
`Synthesis of Gastrins
`
`The main difficulties encountered in the synthesis of gastrin
`
`and CCK related peptides derive from those structural elements
`
`which are essential for the biological activity and thus, di—
`
`rectly involved in the mechanism of action of this class of hor—
`
`mones, i.e.
`
`from the thioether function of methionine,
`
`the in—
`
`dole side—chain function of tryptophan and the tyrosine—O-sul—
`
`fate moiety. This sequence—dependent enhanced reactivity par-
`
`ticularly of the methionine and tryptophan residue towards
`
`oxidants and electrophiles as well as the instability of the
`
`sulfate—ester function has represented for years a challenge
`
`for the peptide chemist.
`
`
`Oxidation of methionine: The pronounced tendency of the
`
`methionine residues in the gastrins and CCK—peptides to oxi-
`
`dation to the corresponding S—oxide derivatives with concomi—
`
`tant loss of activity (16 —17)
`
`implies careful avoidance of
`
`traces of oxidants, e.g. peroxides and heavy metals along the
`
`various steps of synthesis and purification as well as of air—
`
`oxygen to prevent desactivation. Even if reduction of S-oxides
`
`with thiols is possible (18), handling of these peptides for
`
`in Vitro and in vivo assays becomes delicate.
`
`-257-
`
`-257-
`
`
`
`
`

`

`258
`
`Additionally the lability of the tyrosine-O—sulfate ester
`
`prevents an "a posteriori reduction" of S-oxide derivatives
`
`by thiols (15). Basing on first indications of Morley (l7),
`
`Kenner et al.
`
`(19) and Wfinsch et al.
`
`(5 - 6) both independently
`
`revealed the possibility to replace the methionine residue
`
`in the gastrins by the branched aliphatic side-chain of
`
`leucine with retainment of full biological activity. Subsequent
`
`substitutions of methionine by norleucine or its oxa-analogue
`
`methoxinine proved to be equally efficient
`
`(20).
`
`The related
`
`analogs were equipotent to the parent methionine— hormone in
`
`stimulating gastric acid secretion.
`
`Oxidative degradation of tryptophan: Since the first studies
`
`on tryptOphan—containing peptides it is known that the indole
`
`function undergoes facile oxidation with production of
`
`usually colored complex mixtures consisting of components
`
`which are not yet studied in detail. This oxidative
`
`degradation occurs in pronounced manner in acid solutions,
`
`e.g. as needed in acidolytic deprotection steps.
`
`It has
`
`been, however, observed that addition of phenols to free
`
`the acid mixtures from halogens as well as operating in inert
`
`atmosphere such as under argon or adding antioxidants, e.g.
`
`diethylphosphite or preferentially thiols, constitute effi-
`
`cients methods to prevent or at least to minimize the oxi—
`
`dative destruction of tryptophan residues.
`
`Alkylation of tryptophan: A more serious side reaction at the
`
`level of tryptophan residues was detected in our laboratory
`
`during the synthetic work on gastrins, i.e.
`
`the alkylation
`
`of the indole ring as generated in the acidolytic removal
`
`of protecting groups. First indications of a possible chemical
`
`modification of the indole group derived from the synthetic
`
`studies on glucagon (21) and then, particularly on 15—leucine—
`
`human little gastrin-I (6).
`
`An exact identification of the side products and thus, of the
`
`nature of the side reaction which had occurred, was difficult
`
`because of the presence of two tryptophan residues in the
`
`little gastrin molecule. We
`
`then succeeded to identify this
`
`side reaction during the synthesis of a leucine-analogue of
`
`-258-
`
`-258-
`
`
`
`
`

`

`259
`
`minigastrin-I(10)initially prOposed to correspond to the
`
`C-terminal tridecapeptide amide of little gastrin-I(22).
`
`Upon removal of the protecting groups on tert-butanol basis
`
`by exposure to trifluoroacetic acid and partition chromato-
`
`graphy of the resulting crude product on Sephadex G—25,
`main side fraction was identified as 10—Nin-tert-butyl—
`
`the
`
`tryptophan-11—leucine-minigastrin—I by mass- and nmr-
`
`spectroscopy of the C-terminal chymotryptic fragment.
`
`Subsequently in collaboration with Kisfaludy and associates,
`
`studies on model compounds allowed the characterization of
`
`the various alkylation products of the indokering formed in
`
`the acidolytic cleavage of protecting groups on tert-butanol
`basis. Besides the Nin-tert-butyl—derivative, additionally
`
`mono-C-tert—butyl-, poly-C-tert-butyl- and poly—C—, N-tert-
`
`butyl—derivatives are produced whereby the extent of such
`
`alkylations and the distribution pattern of the various
`
`substitutions at the indokafunction were found to strongly
`
`depend on the acids used, on time and temperature (23). To
`
`possibly minimize this deleterious side reaction which causes
`
`remarkable loss
`
`of product,
`
`the use of scavangers, e.g.
`
`indole derivatives, ethers and thioethers as well as a
`
`series of thiols and their mixtures, have carefully been
`
`examined by extensive comparative studies. All efforts to
`
`entirely suppress indole alkylation failed, even if
`
`1,2—ethanedithiole was found to efficiently reduce the rate
`
`of this side reaction as shown in Fig.
`
`2
`
`(11).
`
`Fortunately various analytical methods e.g.
`
`1 H-nmr, tlc,
`
`hplc, were found to be sufficiently sensitive to permit
`
`detection and quantitative monitoring of the extent of such a
`
`side reaction,
`
`thus remarkably facilitating the purification
`
`processes.
`
`In this context noteworthy is the observation
`
`that the rate of alkylation of the indole function strongly
`
`depends on the chemical environment of the tryptophan
`
`residue. While during the synthesis of gastrins and gastrin
`
`analogs as well as gastrin—related peptides tert-butyl—
`
`tryptophan-derivatives have been detected in extents up to
`
`15 % even applying efficient scavangers (11), no alkylation
`
`-259-
`
`-259-
`
`
`
`
`

`

`260
`
`A260
`
`28min
`
`A290
`
`O
`
`8
`
`12
`
`16
`
`20
`
`24
`
`Fig. 2.
`
`lec of crude 1-des-tryptophan, 11-leucine-minigastrin-I
`
`as resulting from exposure of the fully protected
`tridecapeptide derivative Boc-Leu—[Glu(OBut )]5-A1a-
`Tyr(But ) -Gly-Trp-Leu-Asp(OBut)--Phe—NH2 to I)
`trifluoroacetic acid/anisole, 10:1 and II)
`
`trifluoro—
`
`acetic acid/anisole/1,2-ethanedithiol, 10:1:0.25.
`
`Chromatographic conditions: u-Bondapak C 18
`
`(30 x 0.4 cm); eluents: A) 0.1 M sodium phosphate,
`
`pH 5.4;
`
`B) acetonitrile/eluent A, 45:55;
`
`linear
`
`gradient of 35 % B to 80 % B in 30 min;
`
`flow rate:
`
`1.7 ml/min
`
`of the tryptOphan residue was observed upon acidolytic
`
`removal of tert—butanol and 1—adamantanol derived protecting
`
`groups from fully protected somatostatin (24). Consequently,
`
`this sequence-dependent reactivity to electrophiles strongly
`
`questions the general efficacy of acidolytic deprotection
`
`procedures devised on the basis of model studies with simple
`
`tryptophan-compounds.
`
`Racemization:
`
`An additional serious difficulty encountered
`
`in the synthesis of gastrins is related to the last fragment
`
`condensation step, i.e. assembly of the N—terminal pentapeptide
`Pyr-Gly—Pro-Trp-Leu-OH with H—[Glu(OBut)]5-Ala-Tyr(But)-Gly—
`
`-260-
`
`-260-
`
`
`
`
`

`

`2m
`
`Trp~X—Asp(OBut)—Phe-NH2 via DCC/HONSu even using a 3-to 4-fold
`excess of the carboxyl component (5). The
`low acylation rate
`
`induced us to a detailed investigation (11) of various known
`
`coupling methods (Table I).
`
`With benzotriazole as additive the acylation rate was sensibly
`
`enhanced; concomitantly quantitative racemization of the
`
`C—terminal
`
`leucine residue occurred (25). This coupling procedure
`
`proposed by Konig and Geiger
`
`(26) has widely been used and it is
`
`surprising that its negative side effects hmxanot been recognized
`
`earlier. The danger of related remarkable racemization rates
`
`has been confirmed by Sieber et al.
`
`in the synthesis of insulin
`
`(27) and by us in the synthesis of secretin (28) and PHI
`
`(29).
`
`The experiments listed in Table I indicate as only
`
`alternative to the DCC/HONSu method (30)
`
`the use of
`
`2-morpholinoethylisocyanide in presence of N-hydroxysuccinimide
`
`(31).
`
`These above summarized meticulous studies on the observed side
`
`reactions as well as the experiences gained in parallel
`
`synthetic studies on other gastrointestinal hormones allowed
`
`an efficient feedback for an optimization of the synthetic routes
`
`to gastrins-I (11)
`
`(Fig. 3) and gastrin-I-related peptides (12)
`
`(Fig. 4).
`
`The desired peptides became accessible at a
`
`high degree of purity as judged by thin—layer chromatography
`
`under various conditions, high pressure liquid chromatography
`
`in optimized conditions, electrophoresis, uv- and nmr—
`
`spectroscopy, amino acid analyses of the acid and enzymatic
`
`hydrolysates as well as by chiral analysis according to the
`
`method of Bayer and associates (32).
`
`Synthesis of tyrosine-O-sulfate peptides
`
`As mentioned initially the closely related gut hormones CCK
`
`and gastrin-II share as common characteristic a tyrosine-O-sulfate
`
`residue, whereby for latter hormone the nonsulfated form-I has
`
`also been isolated. The question arises of whether circulating
`
`in plasma the gastrins are sulfated and desulfation takes
`
`place during isolation or of whether both the sulfated and
`
`nonsulfated gastrins constitute native components. To shed
`
`light on this undoubtedly important question synthetic
`
`-261-
`
`-261-
`
`
`
`
`

`

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`

`

`264
`
`Z-Glu(OBu’)-Ala-Tyr(Bu’)-Gly-OH[10-13] + H-Trp-Nle-Asp(OBu’)—Phe-NH2 [Nle15][l4-17b]
`
`DCC/HONSu
`
`Z-Glu(OBu')~Ala-Tyr(Bu')-Gly-Trp-Nle-Asp(0BuU-Phe—NH; [N181 5 n 10—17“
`
`Hde
`
`Z-[Glu(OBu‘)|4-OH[6-—9] + H-Glu(OBu’)—Ala-Tyr(Bu’)-Gly-Trp-Nle-Asp(0Bu’)-Phe-NH2 [Niel Sum—171)]
`
`DCC/HONSu
`
`Z-[Glu(OBu’)ls-Ala-Tyr(Bu’)-Gly-Trp—Nle-Asp(OBu’)-Phe-NHz [Nlel 5 "6—1721
`
`Hz/Pd
`
`l:VGlu-Gly-Pro-l'rp-Leu-OHl1—5| + H-[Glu(0Bu')15-Ala-Tyr(Bu’)-Gly-Trp—Nle-Asp(OBu’)-Phe-NH2 [Nle15II6—l7b]
`
`DCC/HONSu
`
`Qlu-Gly-Pro-Trp-Leu-[Glu(OBu’)|5-AIa-Tyr(Bu’)-GIy-Trp-Nle-Asp(OBu’)-Phe-NH2 [Nlel 5 H l ——1 7a]
`
`CFacozfl/anisole/ 1 , 2-ethanedithiol
`partition-chromatography
`
`Q1u-Gly-Pro-Trp-Lcu-Glu-Glu-Glu~Glu-Glu-Alu-Tyr-Gly-Trp-Nlc—Asp-Phc-Nl12
`
`[Nlc'l 5 || | ‘ 17b |
`
`Fig. 4. Synthetic route to 15-norleucine—human little
`
`gastrin-I. The identical synthetic scheme was
`
`followed for the preparation of the 15-leucine-
`
`and 15—methoxinine-analog
`
`sulfated gastrins should be accessible.So far,
`
`the synthesis
`
`of sulfated gastrins has not been achieved; previous attempts
`
`in Kenner's laboratory (33) based on direct sulfation of
`
`gastrin using pyridine/SO3 failed. Surprisingly more success
`was obtained with this method for the preparation of CCK-
`
`peptides upon optimization of the reaction conditions and
`
`extensive purification of the final compounds (34 — 35).
`
`Nonetheless several side reactions such as nuclear sulfonation
`
`of the tyrosine side-chain could not be prevented;
`
`the extent
`
`of sulfonation was found to strongly depend on sulfating
`
`agents used, on the operating conditions and on the chemical
`
`-264-
`
`-264-
`
`
`
`
`

`

`265
`
`environment of the tyrosyl residue (36). The mentioned
`
`difficulties compelled us to devise a new synthetic route
`
`to tyrosine—O—sulfate-peptides based on the direct use of
`
`suitable derivatives of tyrosine-Onsulfate. This key inter-
`
`mediate was obtained by sulfation of Z—Tyr-OH with pyridine/
`
`803 followed by its isolation as barium salt. The homogeneity
`of this starting material
`is easily assessed by chromato-
`
`graphic and spectroscopic methods: free tyrosine or sulfonated
`
`derivatives show red—shifted uv-maxima of strong intensities
`
`in basic solution. Equally sensitive is the detection of such
`contaminants by 1H-nmr
`(13). The usefulness of Na—acyl—
`
`tyrosine—O—sulfate as starting material in the synthesis of
`
`peptides was then investigated on the C-terminal sequence
`
`24-33 of CCK (Fig. 5). For this purpose Fmoc-Tyr(SO3Ba1/2)-OH
`was prepared and coupled to the C-terminal hexapeptide
`
`derivative as outlined in the synthetic scheme; subsequent
`
`removal of the Na—protecting group with piperidine proceeded
`
`smoothly as well as the N-terminal elongation to the deca-
`peptide derivative 24-33, which upon Na-deprotection by
`treatment with piperidine represents a suitable key-
`
`fragment for the synthesis of higher molecular weight
`
`CCK-peptides. Removal of the residual acid-labile protecting
`
`groups by exposure to 90 % trifluoroacetic acid followed
`
`by chromatographic purification of the crude product led
`
`to the CCK-decapeptide in over 60 % yield as homogeneous
`
`material as judged by all the analytical assays performed
`
`(14). The sulfate-ester moiety was found to be surprisingly
`
`stable towards fikzacidolytic deprotection step, most
`
`probably because of the sequential proximity of the strong
`basic function of the arginine side-chain.
`In fact the sulfate
`
`ester function in tyrosine-O—sulfate and related peptides
`is hydrolysed under identical conditions usually to about
`20 %. As expected the alkylation of the tryptophan
`
`residue and the high tendency of the methionine residues
`
`to oxidation were again observed. Unfortunately reduction
`of
`the S—oxides with thiols as well as partial suppression
`of indole-alkylation by thiols as scavangers is precluded
`by the facile hydrolysis of the sulfate ester under acidic
`
`conditions in the presence of thiols (15).
`
`-265-
`
`-265-
`
`
`
`
`

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`267
`
`Thus, as for the gastrins the search for stable
`
`analogs
`
`of CCK-peptides became important leading us to the
`
`design of two nonapeptides corresponding to the C—terminal
`
`COR-sequence 25—33 in which methionine-28 was replaced by
`
`threonine — in analogy to caerulein,
`
`the CCK-active peptide
`
`of amphibian skin -
`
`and methionine—31
`
`by leucine and
`
`norleucine, respectively. The syntheses of the two analogs
`were again successfully performed using Na—acyl—tyrosine—O-
`
`sulfate as intermediate and the desired nonapeptides were
`
`obtained according to the scheme outlined in Fig.
`
`6 as
`
`analytically homogeneous products in satisfactory yields (15).
`
`In vivo and in vitro assays revealed the norleucine—analog
`
`as active as CCK—8 and caerulein, whereas replacement of
`
`methionine by leucine was found to lower the hormonal
`
`potency by a factor of 10. These findings surprisingly differ
`
`from those related to the gastrins and may be explained by
`
`the need of a well balanced amphiphilicity which is
`
`better achieved with the combination threonine—norleucine
`
`according to the hydrophobic/hydrophilic increments of the
`
`various residues using the factors of Tanford (37). The observed
`high stability of Thr28, Nle31-CCK-[25-33] both in solution
`
`and on storage as lyophilized powder, resulting from the salt
`
`bridge between the O-sulfate-and guanido—function(15,16), its
`
`fast conversion in vitro to the corresponding CCK-octapeptide form
`
`(38) as well as its equipotency to CCK—8 and caerulein (15),
`
`revealed this analog as perfectly suited for physiological and
`
`clinical research.
`
`The experiences gained in these synthetic studies on CCK-
`
`peptides have opened the way to the preparation of sulfated
`
`gastrin-peptides.
`
`Immunochemical studies
`
`The pronounced tendency of the methionine residue to oxidation
`
`and of tryptophan to oxidative degradation may lead to
`
`deleterious chemical modifications of the gastrin molecule
`
`during its labelling with radioactive iodine unter oxidative
`
`conditions as needed for the preparation of tracers.
`
`These structural modifications
`
`of
`
`the C—terminal
`
`-267-
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`-267-
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`

`269
`
`sequence of the gastrins undoubtedly arise severe questions
`
`at least in respect to the quantitative interpretation of
`
`data derived from conventional radioimmunoassays. Regarding
`
`the problem methionine a comparative analysis of various
`
`related gastrin analogs revealed the 15-methoxinine-little
`
`gastrin—I — an analog stable to oxidants - as immunoreactive
`
`as the parent methionine peptide towards a series of anti—
`
`gastrin—antisera (20) and thus, suited for the preparation of
`
`tracers. The main difficulty, however, derives from the
`
`tryptophan residue. A systematic investigation of the
`
`oxidative destruction of tryptophan during radioiodination
`
`of tyrosine via the various proposed procedures revealed
`
`that under all conditions employed tryptophan is degraded
`
`to large extents (39 — 40). Formylation of the indole function
`
`was found to prevent the side reaction (40); but even if
`promising in this respect,
`the Nin-formyl group is certainly
`
`not in all cases the most adequate bypass, since its removal
`
`occurs under alcaline conditions where not all peptide
`
`sequences are sufficiently stable.
`
`These shortcomings of the preparation of radioiodinated
`
`tracers,
`
`the molecular heterogeneity of the gastrin—CCK family
`
`and the relatively high immunogenicity of the common C—terminal
`
`sequence as resulting from immunization with conventionally
`
`produced antigens, make the characterization of gastrin-and
`
`CCK—immunoreactivity by the usual
`insufficient.
`
`immunological methods
`
`Our approach to improve selectivity and specificity of these
`
`methods is outlined in Fig. 7. It is based on the incorporation
`
`of a reactive anchor-group via a spacer, at a definite position
`
`of the peptide-chain,
`
`to be used for the specific conjugation
`
`of the peptide factor to high molecular weight carriers as
`
`needed for immunization experiments, as well as for the
`
`incorporation of the tracer group. For this purpose the
`
`maleimido group was selected since it is known to react
`
`rapidly and rather specifically with thiols.
`
`In a first set of experiments maleimido-B-alanine—N—hydroxy-
`
`succinimido ester (41) was used to acylate human little
`
`gastrin—I—[2—17]
`
`(Fig. 8). The resulting gastrin derivative
`
`-269-
`
`-269-
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`2H
`
`Mal—B—Ala—ONSu
`
`+ H—Gly—Pro—Trp—Leu-(Glu) S—Ala—Tyr—GIy—Trp—Met—Asp—Phe—N H 2
`
`DMF / ACN
`
`Mal—B—Ala—GIy—Pro-Trp—Leu— ( Glu) 5—AIa—Tyr—Gly—Trp—Met—Asp—Phe-NH 2
`
`peptide content (Mr = 2138.4)
`
`: 90 %
`
`hplc (u—Bondapak C18):
`
`isocratic elution with acetonitrile/0.1 M sodium phosphate
`
`(pH 5.0), 28:72, flow rate 1.4 ml/min, 280 nm;
`
`tlc: n-BuOH/ACOH/Pyridine/H20; 60:6:20:2H
`
`n—BuOH/AcOH/H20;60:flh20
`
`Fig. 8. Synthesis of maleoyl-B-alanyl—human little
`
`gastrin-I-[2-17]
`
`was found to be well suited for a smooth and clean preparation
`
`of radiolabelled tracers via its reaction with {125 II-desamino-
`tyrosyl-cysteine,
`[3H]-acetylcysteine or [3SS]-cysteine;
`the
`
`related derivatives were found to retain full immunoreactivity,
`
`e.g. Fig. 9. With this approach the problem related to the
`
`methionine and tryptophan residue would be resolved.
`
`However,
`
`to bypass also
`
`radiolysis of the gastrin molecule
`
`and thuslthe storage problem as well as waste-problems,an
`
`alternative fluorescence—immunoassay based on a fluorogenic
`
`enzyme substrate linked to the hormone molecule (Fig. 10) was
`
`elaborated. The fluorogenic tracer was used to develop a double-
`
`antibody enzyme-immunoassay for gastrin which showed to be
`
`sufficiently sensitive to determine normal gastrin levels in
`
`healthy humans
`
`(41).
`
`The conventional conjugation procedures used in the preparation
`
`of antigens employ reagents, e.g. carbodiimides, which undoubtedly
`
`involve the most reactive functions along the hapten molecule,
`
`e.g.
`
`in the case of gastrins the various carboxyl functions in
`
`the central region of the molecule. This fact may be responsible
`
`for the observed high immunogenicity of the C-terminal
`
`-271-
`
`-271-
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`

`

`272
`
`O.
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`110
`
`[3/80x100
`
`0
`
`10
`
`pmol/l
`
`10
`
`Fig. 9. Comparative immunoreactivities of HG-17 (o——o——o)
`
`and of the conjugate cysteine/Mal—B-Ala-HG—[2—17]
`1251-HG—[1-17]
`
`in cross reactions versus
`
`(o—~o——o)
`
`using antigastrin-antiserum No. 2127 of Rehfeld
`
`tetrapeptide and thus, for the difficult production of
`
`gastrin— or CCK—specific antisera (42). On the assumption that
`
`selectivelinkage of the gastrin at its N-terminus to carriers
`
`with concomitant full exposure of the unmodified hapten
`
`molecule to the antigen recognition may lead to more selective
`
`and specific antisera, maleimido—B-alanyl—human little gastrin—
`
`[2—17] was conjugated to reduced RNase A. The antisera raised
`
`in rabbits with this antigen were used to titrate the immuno—
`
`reactivity of the gastrin sequence. As shown in Fig. 11, a
`
`sharp cooperative transition was observed upon incorporation
`
`of the third glutamic acid residue, while the C-terminal
`
`tetrapeptide is recognized to very low extents. A similar
`
`sigmoidal curve in function of chain—length of the gastrin
`
`was determined for the acid secretion potency (12) as
`
`shown in Fig. 12 and for the onset of the proposed "active"
`
`B-structure as determined by circular dichroism measurements
`
`-272-
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`100
`
`75
`
`
`
`1C50(HGl-17)/IC50x100
`
`1
`
`3
`
`5
`
`7
`
`9
`
`11
`
`13
`
`15
`
`T7
`
`Pyr-Gly—Pm-Trp—Leu-Glu—Glu—Glu-GIu-Glu-Ala—Tyr—Gly-Trp-Met-Asp-Phe-NH2
`pyr____.—___‘3‘:— MHz
`H-Gl y—————————~—————Me1 —Phe-NH2
`
`Pyr-——————- Nle —— Phe-NH2
`
`Pyr
`Nle -— Phe-N H2
`Pyr—————— Nle —Phe-NH2
`
`Pyr——-——— Nle
`Phe-NHZ
`Pyr,———- Nle———— Phe-NH2
`
`Fig. 11. Titration of the immunoreactivity of the human
`
`little-gastrin-I sequence
`
`100
`
`onO
`
`“/aAktivitdt mC
`
`1.0
`
`20
`
`123 I. 567891011121314151617
`
`{élu—Gly —Pro -Trp - Leu-Glu -Glu—Glu- Glu-Glu—Alu -Tyr-Gly - Trp-Nle-Asp- Phe-NH2
`
`H-Leu ————————————————————Phe-NH2
`
`félu ———————————————Phe~NH2
`
`fism——————————_Phe-NH2
`
`12am ———————————Phe—NH2
`
`is
`
`I
`
`v
`
`A
`
`o
`
`FGIu——._—Phe—NH2 a
`
`félu—————_Phe— NH2
`
`H-Trp—-—Phe- NH2
`
`A
`
`0
`
`Fig. 12. Stimulation of gastric acid secretion in rats by
`
`gastrin-I-peptides expressed as percentage of the
`response to Nle15—HG—17
`
`-274-
`
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`
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`

`

`275
`
`of the gastrin peptides in trifluoroethanol
`
`(43 — 44).
`
`This parallel development of the conformational, biological
`
`and immunological properties in function of chain—length
`
`strongly suggests that the immunodeterminant expressed by
`
`this new approach may possibly be conformation-dependent.
`
`This should be equivalent to an enhancement of selectivity
`
`and specificity of the antisera. Further experiments related
`
`to both the gastrins and cholecystokinins are in progress
`
`to confirm these promising findings.
`
`References
`
`1.
`
`Tracy, H.J. and Gregory, R.A.
`
`(1964) Physiological
`
`properties of a series of synthetic peptides
`
`structurally related to gastrin I. Nature, 204, 935—938.
`
`Gregory, H., Hardy, P.M., Jones, P.S., Kenner, G.W.
`
`and Sheppard, R.C.
`
`(1964) The antral hormon gastrin;
`
`structure of gastrin. Nature, 204, 931-933.
`
`Gregory, R.A. and Tracy, H.J.
`
`(1966) Isolation of two
`
`gastrins from human antral mucosa. Nature, 209, 583.
`
`For review see Mutt, V.
`
`(1983) Chemistry of the
`
`Gastrointestflwl Hormones and Hormone-like Peptides and
`
`a Sketch of Their Physiology and Pharmacology. Vitamins
`
`and Hormones. Vol. 39, 231-427.
`
`Wfinsch, E. and Deimer, K.H.
`
`(1972) Zur Synthese des
`
`[15-Leucin]Human-Gastrin I. II. Mitteilung: Herstellung
`
`der Gesamtsequenz. Hoppe-Seyler's Z. Physiol.Chem. 3E3,
`1255—1258.
`
`Wfinsch, E., Jaeger, E., Deffner, M. and Scharf, R.
`
`(1972)
`
`Zur Synthese des [15-Leucin]Human-Gastrins I. III. Mitteilung:
`
`Zur Reindarstellung des synthetischen Heptadecapeptidamids.
`
`Hoppe-Seyler's Z. Physiol.Chem. 3E3, 1716-1720.
`
`Wfinsch, E., Wendlberger, G., Hallet, A., Jaeger, E.,
`
`Knof, S., Moroder, L., Scharf, R., Schmidt, 1.,
`
`Thamm, P. and Wilschowitz, L.
`
`(1977)
`
`Zur Totalsynthese
`
`des Human—Big—gastrins I und seines 32—Leucin—Analogons.
`
`Z. Naturforsch. 32c, 495—506.
`
`-275-
`
`-275-
`
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`

`

`276
`
`Wfinsch, E., Wendlberger, G., Mladenova-Orlinova, L.,
`
`Gehring, W., Jaeger, E., Scharf, R., Gregory, R.A.
`
`and Dockray, G.J.
`
`(1981) Totalsynthese des Human-
`
`Big—Gastrins I, Revidierte Primarstruktur. HOppe—Seyler's
`
`Z. Physiol. Chem. 362, 179-185.
`
`Moroder, L., Dress, E., Jaeger, E. and Wfinsch, E-
`
`(1978)
`
`Zur Synthese von [11—Leucin]Human—Minigastrin I. Darstellung
`
`der Gesamtsequenz. Hoppe-Seyler's Z. Physiol. Chem. 359,
`
`155—164.
`
`10.
`
`Jaeger, E., Thamm, P., Schmidt, J., Knof, S., Moroder, L.
`
`and Wfinsch, E.
`
`(1978)
`
`Zur Synthese von [11-Leucianuman—
`
`Minigastrin I. II. Mitteilung: Reindarstellung des
`
`synthetischen Tridekapeptidamids sowie Isolierung und
`
`Strukturaufklarung eines Synthese-Nebenprodukts.
`
`Hoppe—Seyler's Z. Physiol.Chem. EEE, 155—164.
`
`11.
`
`Moroder, L., Gehring, W., Nyfeler, R., Scharf, R.,
`
`Thamm, P. and Wendlberger, G.
`
`(1983)
`
`Zur Synthese von
`
`Human—Little—Gastrin I und dessen Leucin-15, Norleucin-15
`
`und Methoxinin—15—Analoga. HOppe—Seyler's Z. Physiol.
`
`Chem. Egg, 157—171.
`
`12.
`
`thring, W., Moroder, L., Borin, G., Lobbia, A., Bali, J.P.
`
`and Wfinsch, E.
`
`(1984)
`
`Synthese von Gastrinaktiven
`
`Peptiden. Untersuchungen zur Struktur—Wirkungsbeziehungen
`
`des natfirlichen Hormons Human-Little—Gastrin I. Hoppe-
`
`Seyler's Z. Physiol. Chem. EQE, 33*94-
`
`13.
`
`14.
`
`Moroder, L., Wilschowitz, L., Jaeger, E., Knof, S.,
`
`Thamm, P. and Wfinsch, E.
`
`(1979)
`
`Synthese von Tyrosin-O-
`
`sulfat—haltigen Peptiden. HOppe—Seyler's Z. Physiol. Chem.
`
`ggg, 787-790.
`
`Wfinsch, E., Moroder, L., Wilschowitz, L., Géhring, W.,
`
`Scharf, R. and Gardner, J.D.
`
`(1981)
`
`Zur Totalsynthese
`
`von Cholecystokinin-Pankreozymin. Darstellung des
`
`verknfipfungsféhigen "Schlfisselfragments" der Sequenz
`
`24—33. Hoppe—Seyler's Z. Physiol. Chem. EQE, 143—152.
`
`-276-
`
`-276-
`
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`277
`
`15. Moroder, L., Wilschowitz, L., Gemeiner, M., Gohring, W.,
`
`Knof, S. Scharf, R., Thamm, P., Gardner, J.D., Solomon, T.E.
`
`and Wfinsch E.
`
`(1981)
`
`Zur Synthese von Cholecystokinin-
`
`Pankreozymin. Darstellung von [28-Threonin, 31-Norleucin]-
`
`und [28-Threonin, 31-Leucin] Ch