J. Peptide ReJ. 50, /997, 222-230
`Printed in the U,rited Statts of America - all right.\' resen·ed
`
`Copyright© Munksgaard, 1997
`JOURNAL OF PEPTIDE RESEARCH
`ISSN /397-002X
`
`Synthesis, biological activity and isomerism of guanylate
`cyclase C-activating peptides guanylin and uroguanylin
`
`JOACHIM KLODT, 1 MICHAELA KUHN, 1 UTE C. MARX, 1•2 SILKE MARTIN, 1 PAUL ROSCH,2
`WOLF-GEORG FORSSMANN 1 and KNUT ADERMANN 1
`
`1Niedersiichsisches /11stitutfar Peptid-Forschung (/PF), Hannover, and
`2 Lehrstuhlfiir Struktur und Chemie der Biopolymere, Universitiit Bayreuth, Germany
`
`Received 12 February, revised 19 March. accepted for publication 10 May 1997
`
`Recently, the peptides guanylin and uroguanylin were identified as endogenous ligands of the membrane-bound
`guanylate cyclase C (GC-C) that is mainly expressed in the intestinal epithelium. In the present study, bioactive
`guanylin and uroguanylin have been prepared by solid-phase methodology using Fmoc/HBTU chemistry. The
`two disulfide bonds with relative 1/3 and 2/4 connectivity have been introduced selectively by air oxidation of
`thiol groups and iodine treatment of Cys(Acm) residues. Using this strategy, several sequential derivatives were
`prepared. Temperature-dependent HPLC characterization of the bioactive products revealed that guanylin-related
`peptides exist as a mixture of two compounds. The isoforms are interconverted within approximately 90 min,
`which prevents their separate characterization. This effect was not detected for uroguanylin-Iike peptides. Synthetic
`peptides were tested for their potential to activate GC-C in cultured human colon carcinoma cells (T84), known
`to express high levels of GC-C. The results obtained show that both disulfide bonds are necessary for GC-C
`activation. The presence of the amino-terminally neighboring residues of Cysl04 for guanylin and CyslO0 for
`uroguanylin has been found to be essential for GC-C stimulation. Unexpectedly, a hybrid peptide obtained
`from substitution of the central tripeptide AYA of guanylin by the tripeptide VNV of uroguanylin was not
`bioactive. © Munksgaard 1997.
`
`Kev words: cGMP: disulfide: guanylate cyclase C: guanylin: uroguanylin; peptide hormone; peptide synthesis;
`topological isomerism
`
`Abbreviations: Acm, acetamidomethyl: Boe. rerr-butyloxycar(cid:173)
`bonyl; cGMP, cyclic 3',5'-guanosine monophosphate: DCM.
`dichloromethane; DIEA. N.N-diisopropylethylamine: EDT.
`ethanedithol; ESMS, electrospray mass spectrometry: Fmoc.
`fluorenylmethoxycarbonyl: GC-C. guanylate cyclase C: HBTU.
`2-( 1 H-benzotriazole-l-yl)- l .1.3.3-tetramethyluronium hexafluoro(cid:173)
`phosphate; HOAc. acetic acid: HOBt. N-hydroxybenzotriazolc:
`lsc, short-circuit current: MeCN. acetonitrilc: NMP. N-methyl(cid:173)
`pyrrolidinone; NMR, nuclear magnetic resonance: PHB. p-a\koxy(cid:173)
`bem:yl alcohol; TBME, tert-butylmethylether: TBTU. 2-( lH-benzo(cid:173)
`triazole-1-ylJ-l, 1,3,3-tetramethyluronium tetrafluoroborate: TFA.
`trifluoroacetic acid.
`Residue numbers refer to the circulating proguanylin-(22-115)
`(7) and cDNA-deduced uroguanylin precursor containing I 12
`amino acids (11) and are used throughout the text.
`Part of this work has been presented in abstract form at the 4th
`International Symposium, Solid Phase Synthesis & Combinato(cid:173)
`rial Chemical Libraries, Edinburgh, Scotland [Adermann. K.. Neitz.
`S., Marx, U., Rosch. P. & Forssmann, W.G. (1995) Abstract Pl].
`
`Guanylin and uroguanylin are novel mammalian pep(cid:173)
`tide hormones that are ligands of intestinal receptor
`guanylate cyclase C (GC-C). GC-C activation increases
`the intracellular concentration of cyclic guanosine
`monophosphate ( cGMP), which in tum modulates in(cid:173)
`testinal water and electrolyte secretion (1-5). The en(cid:173)
`docrine role of this new family of regulatory peptides
`with respect to epithelial ion transport and a functional
`link between intestine, kidney and other tissues are top(cid:173)
`ics of current research in physiology. Guanylin, origi(cid:173)
`nally isolated as a peptide of 15 residues from rat
`jejunum (6), also circulates in human plasma as a
`larger hormone containing 94 amino acids (7).
`Uroguanylin was isolated from urine and represents a
`second activator of GC-C (8, 9). Recently, an
`amino-terminally elongated form of 24 amino acid resi(cid:173)
`dues was shown to represent a plasma-circulating form
`of uroguanylin (I 0, 11 ). It was also reported that
`prouroguanylin is present in plasma and urine in pa(cid:173)
`tients with chronic renal failure (12).
`
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`Both guanylin and uroguanylin share a significant
`sequence homology with a specific family of bacterial
`enterotoxins (Fig. 1). In particular, Escherichia coli
`heat-stable enterotoxin STa causing secretory diarrhea
`in mammals was shown to activate GC-C with a sig(cid:173)
`nificantly higher efficacy than guanylin and uroguany(cid:173)
`lin (13, 14). It was predicted that the four cysteine
`residues of guanylin and uroguanylin form two disul(cid:173)
`fide bonds in a connectivity that is also contained in
`E. coli STa ( 15, 16). Synthesis of the three possible dis(cid:173)
`ulfide isomers of guanylin confirmed that only one of
`the several possible disulfide isomers acts as a GC-C
`activator and therefore is able to increase the intracellu(cid:173)
`lar cGMP level (2, 17). Similarly, uroguanylin with
`equivalent disulfide connectivity was shown to stimu(cid:173)
`late GC-C in a concentration-dependent manner (9, 10).
`It is generally accepted that the other two disulfide iso(cid:173)
`mers of uroguanylin are biologically inactive.
`Considering the molecular interaction of guanylin
`and uroguanylin with the extracellular domain of their
`receptor, the disulfide loops drastically reduce their con(cid:173)
`formational flexibility, resulting in a highly con(cid:173)
`strained three-dimensional structure. In this context, a
`unique feature of guanylin and uroguanylin is that both
`peptides exist in two distinct conformations. Although
`not separable by chromatographic methods, it was re(cid:173)
`ported that, for synthetic guanylin-(103-115) and the
`tryptic fragment (94-115) obtained from recombinant
`proguanylin, two isomers are present in aqueous solu(cid:173)
`tion, as detected by NMR spectroscopy (18). In that
`study, it was concluded that one of the two forms adopts
`a conformation similar to that observed for E. coli
`enterotoxin STa (19). We have shown that guanylin-(99-
`115) also appears as a mixture of two compounds which
`are detectable by means of low-temperature HPLC (20).
`Very recently, it was reported that solid-phase synthe(cid:173)
`sis of human uroguanylin-(97-112) yields two topo(cid:173)
`logical isomers which could be separated by HPLC
`(21 ). In another study, it was reported that only one
`of these isomers is bioactive and different native forms
`of uroguanylin have been identified using polyclonal
`antisera raised against the two conformational isomers
`of synthetic uroguanylin (12).
`In this communication, we describe a general strat(cid:173)
`egy that allows solid-phase synthesis of guanylin/
`uroguanylin-type peptides with regioselective introduc(cid:173)
`tion of disulfide bonds. Using this tool, several deriva(cid:173)
`tives of guanylin and uroguanylin have been
`
`Guanylin (99-115)
`Uroguanylin (97-112)
`Uroguanylin (89-112)
`E.coli s·1a
`
`EDPG-I Y
`ND C
`·. N
`FKTLRTIAND . ·
`_·
`NTFY
`
`N_c==c,-
`
`GC-C-activating peptides guanylin and uroguanylin
`
`synthesized to gain insight into sequential and confor(cid:173)
`mational requirements for the interaction between the
`peptide regulators and their receptor.
`
`EXPERIMENTAL PROCEDURES
`
`Reagents for peptide synthesis. Fmoc amino acids
`(L-configuration) were purchased from Orpegen
`(Heidelberg, Germany) and PerSeptive Biosystems
`(Wiesbaden, Germany). Protective groups were:
`Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Acm), Cys(Trt),
`Glu(OtBu), Lys(Boc), Ser(tBu), Thr(tBu), Tyr(tBu).
`Syntheses were carried out using TentaGel resins (Rapp
`Polymere, Ttibingen, Germany). HBTU was obtained
`from Perkin-Elmer/ABI (Weiterstadt, Germany),
`TBTU was from Peboc (Llangefni, Wales). NMP was
`purchased from Merck (Darmstadt, Germany). Other
`reagents and solvents were of analytical or higher grade
`and obtained from Merck or Fluka (Neu-Ulm, Ger(cid:173)
`many). E. coli heat-stable enterotoxin STa was from
`Sigma (Deisenhofen, Germany).
`
`Solid-phase peptide synthesis. Peptide assemblies
`were carried out in NMP at a scale of 0.1 mmol using a
`preloaded Fmoc-Cys(Trt)-TentaGel S Trt or S PHB resin
`(0.23 mmol/g) for guanylin-related peptides and
`Fmoc-Leu-TentaGel S Trt (0.19 mmol/g) for uro(cid:173)
`guanylin-related peptides. For the synthesis of guanylin
`peptides, Cys104 and Cys112 were Acm-protected,
`whereas Cys107 and Cysl 15 were Trt-protected.
`Uroguanylin was synthesized with Trt protection for cys(cid:173)
`teines in positions 103 and 111, and Acm protection
`at CyslOO and Cys108. As a standard procedure, all
`peptides were synthesized on a 433A peptide synthe(cid:173)
`sizer (Perkin-Elmer/AB!) with conditional conductiv(cid:173)
`ity monitoring of Fmoc deprotections. In addition,
`guanylin-(99-115) and uroguanylin-(89-112) were
`synthesized manually on preloaded TentaGel S PHB
`resins (2 mmol) using double coupling cycles for each
`residue (10 mmol Fmoc amino acid, 9.5 mmol TBTU,
`4 mmol HOBt and 20 mmol DIEA). Acylations were
`performed for 2 h and monitored by ninhydrin assay
`(22). Fmoc groups were cleaved with 20% piperidine
`in NMP for 20 min. After completion of syntheses,
`resins were washed with DCM and dried under vacuum.
`For resin cleavage and deprotection, the dry peptidyl
`resins were treated at room temperature with a fresh
`mixture of TFA/EDT/H20 (94:3:3, 20 mL/g) for 1.5-
`2 h. Subsequently, the peptide-containing cleavage cock(cid:173)
`tail was filtered and slowly dropped into chilled TBME
`(100 ml/20 mL TFA). Precipitated crude peptides were
`separated by centrifugation, washed twice with cold
`TBME and dried under vacuum without lyophilization.
`
`FIGURE I
`Primary structures of GC-C-activating peptides. Conserved resi(cid:173)
`dues are shaded.
`
`Disulfide formation and purification. To introduce the
`first disulfide bond by air oxidation, the crude peptide
`was dissolved in water (0.5-1 mg/mL). pH was ad-
`
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`J. Klodt et al.
`
`justed between 7.8 and 8.5 with diluted NH 3• The so(cid:173)
`lutions were stirred until no free thiol was detectable
`by Ellman's reagent (23) and C18-HPLC monitoring.
`Mixtures were acidified with TFA (pH 2.5), loaded di(cid:173)
`rectly onto a preparative Cl8-HPLC column and puri(cid:173)
`fied (Vydac Cl8, Hesperia, CA, 47 x 300 mm, 300 A,
`15-20 µm, buffer A; 0.1% TFA, buffer B: 0.1% TFA
`in MeCN/water, 80:20, gradient 0-100% B in 60 min,
`flow 50 mL/min, UV detection at 230 nm). Fractions
`containing the pure monocyclic intermediate as de(cid:173)
`tected by ESMS and C18-HPLC were collected and
`lyophilized. Cleavage of Acm groups and introduction
`of the second disulfide bond were carried out by dis(cid:173)
`solving the purified monocyclic Acm1-peptides in
`deairized HOAc/0.l M HCl (4:l, pH 2, l mg/mL). A
`solution of iodine (10-20 eq, 0.05 M) in HOAc was
`added and after 30-60 min the excess iodine was re(cid:173)
`duced by the addition of fresh 0.1 :'v1 sodium thiosul(cid:173)
`fate, resulting in a colorless solution. After dilution
`with water (2 volumes), the bicyclic products were
`obtained from preparative C 18-HPLC (Vydac CI 8, 20
`x 250 mm, 300 A, 10 µm, buffer A: 0.1 o/c TFA; buffer
`B: 0.1 % TFA in MeCN/water 80:20, gradient 0-100%
`B in 60 min, flow 8 mL/min, 230 nm). Purification
`was performed at 50 °C (guanylin peptides) or room
`temperature (uroguanylin peptides). In the case of in(cid:173)
`sufficient purity, HPLC purification of bicyclic prod(cid:173)
`ucts was repeated.
`
`Analytical methods. Analytical HPLC was performed
`on a Vydac C 18 column (5 µm, 300 A, 4.6 x 250 mm,
`buffer A: 0.06% TFA; buffer B: 0.05% TFA in 80%
`MeCN, flow rate 0.8 mL/min, 215 nm). Tempera(cid:173)
`ture-dependent HPLC characterization of purified pep(cid:173)
`tides was carried out using an adjustable column oven
`with a Nucleosil Cl8 PPN column (2 x 250 mm, 5
`µm, 100 A, flow rate 0.2 mL/min, 215 nm, Macherey
`& Nagel, Di.iren, Germany). Mass spectrometry was
`performed using a triple-stage quadrupole mass spec(cid:173)
`trometer (Sciex API III, Perkin-Elmer). Peptide
`samples were dissolved in MeCN/water ( 1: l) contain(cid:173)
`ing 0.2% AcOH to a concentration of 0.1 mg/mL and
`infused into the ion spray using a syringe pump at a
`flow rate of 5 µL/min. The ion spray interface was
`set to a positive potential of 5 kV. Amino acid analy(cid:173)
`sis was carried out on a Aminoquant 1090L analyzer
`(Hewlett-Packard, Waldbronn, Germany). Sequence
`analysis was performed on a 494A protein sequencer
`(Perkin-Elmer). Two-dimensional NMR spectroscopy
`(NOESY, Clean-TOCSY, DQF-COSY) was performed
`on a Bruker AMX600 spectrometer at 284 K using stan(cid:173)
`dard methods (24, 25). Synthetic peptides were dissolved
`in H 20/D20 (9: 1) at a concentration of 5-10 mg/mL.
`
`T84 cell cGMP bioassay. Determination of the
`GC-C-stimulating activity of synthetic peptides was evalu(cid:173)
`ated as described elsewhere in detail (1, 7, l 0). Cultured
`
`224
`
`human colon carcinoma (T84) cells were incubated with
`synthetic peptides for 60 min in the presence of the phos(cid:173)
`phodiesterase inhibitor isobutylmethylxanthine (IBMX, I
`mM, Sifma). The peptides were tested in concentrations
`to 10-6 M. The given concentrations refer to
`of 10-
`the net peptide content specified by amino acid analy(cid:173)
`sis. Effects on intracellular cGMP level were compared
`with those of E. coli enterotoxin STa. cGMP was mea(cid:173)
`sured using a specific radioimmunoassay (26).
`
`Ion transport (Ussing chamber experiments). Female
`Wistar rats maintained under standard temperature,
`light conditions and with free access to water and food
`were used. For the assessment of active electrogenic
`ion transport, specimens of proximal colon were dis(cid:173)
`sected and serosal and muscular layers separated. Sheets
`of isolated mucosa were mounted in Ussin& chambers
`(27) with an exposed surface area of 1 cm· and auto(cid:173)
`matically voltage-clamped as described previously (3,
`4). Short-circuit current (lsc) was recorded continu(cid:173)
`ously. After an equilibrium period of 30 min, cumu(cid:173)
`lative concentrations of synthetic peptides were added
`to the luminal side of the colonic mucosa.
`
`RESULTS AND DISCUSSION
`
`Synthesis
`In agreement with earlier observations (2, 2 I) and in
`contrast to other peptides like endothelin (28) and low
`molecular weight toxins (29), oxidative folding of
`tetrathiol-containing precursors of guanylin and
`uroguanylin did not result in bioactive products that
`increase intracellular cGMP concentration. The re(cid:173)
`duced peptides had a poor solubility in aqueous buffers.
`Further attempts to synthesize guanylin with different sets
`of orthogonal side-chain protective groups for the cys(cid:173)
`teine residues like acetamidomethyl (Acm), tert-butyl
`(tBu), tert-butylthio (StBu), p-methoxybenzyl (Mob)
`or allyloxycarbonylaminomethyl (Allocam) resulted in
`products that were only slightly soluble in buffers re(cid:173)
`quired for disulfide formation. To improve solubility,
`guanylin was initially synthesized with additional
`amino-terminal Glu-Asp residues which do not influ(cid:173)
`ence the bioactivity. Guanylin and uroguanylin were
`synthesized using Acm and Trt protection for cysteine
`residues with Cys(Trt) at the C-terminal position. De(cid:173)
`tectable by conductivity monitoring, qualitative ninhy(cid:173)
`drin assay or HPLC/ESMS of intermediates, guanylin and
`uroguanylin represent difficult peptides. Starting from
`Alal 10 (guanylin) and Val106 (uroguanylin), acylations
`and cleavage of Fmoc groups tended to proceed
`slowly. For the manual synthesis of larger amounts of
`peptides, acylations were carried out for 2 h whereas
`Fmoc peptidyl resins were treated for 20 min with pi(cid:173)
`peridine. Exchanging the pairs of cysteine protective
`groups resulted in a product of slightly less purity.
`The first loop between the unprotected cysteines of
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`

`all synthetic compounds was introduced by air oxida(cid:173)
`tion at pH 8. The proceeding oxidation process could
`be monitored easily by Ellman's test (23), HPLC com(cid:173)
`parison of thiol peptides and monocyclic products or
`ESMS analysis after iodoacetamide treatment (30) (Fig.
`2). Depending on the sequence, a period of 12-72 h
`was necessary for this step. For the subsequent work-up
`of monocyclic Acmrpeptides, it was essential to
`acidify the mixture to pH 2.5 (HCl or TFA). As de(cid:173)
`tected by MS and HPLC, direct lyophilization or pu-
`
`(a)
`
`(b)
`
`[M+2H]
`" "
`2+
`
`1373
`
`[M+Ht 1850
`
`2+
`[M+2H] 1372
`
`+
`[M+H] 1848
`
`2+
`[M+2H] 1300
`
`[M+H{ 1703
`
`10
`
`15
`
`20
`
`25
`
`30
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Time (min)
`
`FIGURE2
`HPLC characterization of linear, monocyclic and purified
`peptides. (a) human uroguanylin-(89-112) FKTLRTIAN(cid:173)
`DDCELCVNVACTGCL; (b) human guanylin-(99-115),
`EDPGTCEICAYAACTGC. Top traces, linear peptides after
`TFA cleavage; middle traces, monocyclic Acm2-peptides;
`bottom traces, bicyclic, purified products. HPLC gradient (Vydac
`Cl8): linear and monocyclic peptides, 20-70% buffer Bin 30
`min at room temperature; bicyclic peptide, I 0-50% buffer B in
`40 min at 40 °C. Masses given were determined by ESMS.
`
`GC-C-activating peptides guanylin and uroguanylin
`
`rification of a pH 8 solution of monocyclic peptides re(cid:173)
`sulted in products with a poor solubility, possibly
`caused by further oxidation of cysteines. To obtain fi(cid:173)
`nal products of a high purity, additional HPLC puri(cid:173)
`fications of the monocyclic Acmz-peptides were
`performed. Cleavage of Acm groups with subsequent
`formation of the second disulfide with excess iodine
`at pH 2 (HCl) was a suitable method with suppression
`of disulfide rearrangement (31 ). The absence of HCI
`led to unsatisfactory products. Different solvents like
`mixtures of water, acetic acid and methanol did not
`affect iodine treatment. Alternative attempts at disul(cid:173)
`fide formation using on-resin procedures and the
`solvent-dependent selectivity of iodine treatment of
`free thiols and Acm-protected cysteine residues failed
`(32); dimerization was a significant side effect. Over(cid:173)
`all yields were between 2 and 8% because of the re(cid:173)
`peated HPLC purifications of mono- and bicyclic
`peptides. With guanylin-(99-115) and uroguanylin(cid:173)
`(89-112) as examples, Fig. 2 shows analytical HPLC
`of intermediate compounds and final products which
`were unambiguously characterized by ESMS. Using
`this general synthetic procedure, guanylin and
`uroguanylin as well as several derivatives have been
`prepared and characterized (Table 1 ). As recently com(cid:173)
`prehensively reviewed (31, 33), our results of the syn(cid:173)
`thesis of guanylin and uroguanylin are another
`indication that suitable reaction conditions for each
`single peptide containing multiple disulfide bonds must
`be worked out accurately.
`Unlike other polycyclic peptides with multiple cys(cid:173)
`tines within a short sequence segment, low-tempera(cid:173)
`ture HPLC analysis of purified guanylin-(99-115) (4)
`showed two components in a ratio of about 1 : 1. ESMS
`analysis resulted in only one signal with the expected
`molecular weight of 1703 Da. Reverse-phase HPLC
`with a stepwise increase of column temperature led to
`one sharp absorption at high temperature. Cisltrans
`isomerism of the Aspl00-Prol0l amide bond was
`excluded as the reason for this observation by
`temperature-dependent HPLC analysis of guanylin(cid:173)
`(101-115) (3) with an amino-terminal Pro residue.
`Shown in Fig. 3, a and b, peptides 3 and 4 exhibit the
`identical HPLC characteristic at different temperatures.
`A corresponding temperature effect was observed with
`guanylin-(104-115) (1), [desThrl03]guanylin-(99-
`115) (7) and compounds 12 and 13. In contrast,
`guanylin-(103-115) (2) appears as a single HPLC peak.
`This is consistent with an earlier report (18). Our pre(cid:173)
`liminary NMR experiments with guanylin-(99-115) (4)
`confirmed the presence of two distinct compounds in
`a ratio of about 1: 1 (20). The guanylin/uroguanylin hy(cid:173)
`brid 9 had a major HPLC peak, but two-dimensional
`NMR spectroscopy revealed the presence of two com(cid:173)
`ponents in· equivalent amounts (data not shown). As
`expected, monocyclic Cys(Acm)2 intermediates 10, 11
`and 18 were homogeneous during HPLC. A reversed in-
`
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`2938.5 I l470.5i
`2511.9 / 1257.0i
`2614.1 / 1372.0i
`2469.9 / 1235.0i
`2484.9 / 1242.0i
`2599.0 / 1300.0i
`1667.9 / 1667.5h
`1515.6 / 1516.()h
`1458.7 / 1459.0h
`1845.9 / 1846.0h
`1845.9 / 1846.()h
`1709.9 / )710.0h
`)582.7 / 1582.5h
`)601.8 / 1601.()h
`1573.8 / 1573.5h
`1702.9 / 1703.5h
`1702.9 / 1703.5h
`1458.7 / 1459.0h
`1304.5 I l 3(l4.5h
`I 203.4 / 1203.5h
`
`Calc./ESMS
`Mass (Da)
`
`n.d., not determined.
`i[M+2H]2+.
`h[M+Ht.
`3BiotinAhx, N-(Biotinyl-6-aminocaproyl).
`fSequence from guinea pig uroguanylin obtained by translation of the corresponding mRNA (EMBL database, Accession no. 274738).
`•Monocyclic Aerni-intermediates.
`dSequences from porcine and guinea pig guanylin obtained by translation of the corresponding mRNA (EMBL database, Accession nos. 273607 and 274736.
`cMonocyclic Acm2-intermediates.
`bMonocyclic Acm2-intermediates.
`"Reversed order of disulfide bond formation using Cys(Acm)l07/115 and Cys(Trt)I04/l 12.
`
`n.d.
`Ala 2.13 Arg 0.99 Val 0.96 Mel 0.95 lie 1.00 Leu 3.08 Asx 3.99 Glx 2.35 Gly 1.20 Thr 1.82
`n.d.
`Ala 2.07 Arg 0.99 Val 2.02 Phe 0.97 lie 0.98 Lei 3.05 Asx 4.06 Gly 1.17 Thr 2.21 Lys 0.85
`Ala 2.08 Arg 1.01 Val 1.94 Phe 1.00 lie 0.93 Leu 3.10 Asx 3.09 Glx 0.71 Gly 1.23 Thr 2.57 Lys 1.02
`Ala 2.12 Arg 1.()4 Val 1.94 Phe 0.98 lie 0.98 Leu 3.05 Asx 3.95 Glx 0.71 Gly I. 11 Thr 2.31 Lys 0.97
`Ala 1.04 Val 1.89 Leu 2.(l4 Asx 3.84 Glx 0. 72 Gly 1.19 Thr 0.93
`Ala 3.08 lie 0.98 Asx 0.78 Glx 0.65 Gly 1.03 Thr 1.70 Tyr 0.82 Pro 1.02
`Ala 4.12 lie 0.93 Glx 0.63 Gly 1.15 Thr 0.78 Tyr 0.88 Pro 1.04 Ser 0.45
`n.d.
`n.d.
`Ala 1.0 I lie 0.97 Asx 1.83 Glx 1.42 Gly 2.02 Thr 1.77 Pro 1.02 Val 1.93
`Ala 2.02 lls 0.97 Asx 0.81 Glx 1.31 Gly 4.06 Thr 1.72 Pro 1.06
`Ala 3.06 lie 0. 94 Asx 0.66 Glx I. 12 Gly 2.02 Thr 0. 90 Tyr 0.81 Pro 1.0 I
`Ala 3.22 Ile 0.93 Asx 0.90 Glx 0.72 Gly 1.98 Thr 1.63 Tyr 0.79 Pro 0.98
`Ala :U I Ile 0.95 Asx 0.89 Glx 1.37 Gly 2.23 Thr 1.73 Tyr 0.86 Pro 1.12
`Ala 3.02 lie 0.94 Asx 0.97 Glx 1.56 Gly 2.01 Thr 1.61 Tyr 0.84 Pro 1.08
`Ala 3.31 Ile 1.00 Glx 0.63 Gly 0.99 Thr 1.56 Tyr 0.94 Pro 0.92
`Ala 3.09 lie 0.95 Glx 0.59 Gly I .02 Thr 1.6<) Tyr 0.88
`Ala 3.06 Ile 0.98 Glx 0.68 Gly 1.13 Thr 0.84 Tyr 0.75
`
`20g (BiotinAhx)FKTLRTIANDDCELCVNVACTGCL
`I 9r LQALRTMDNDECELCVNIACTGC
`18° FKTLRTIANDDC(Acm)ELCVNVAC(Acm)TGCL
`17 FKTLRTIANDDCLCVNVAC'TGCL
`16 FKTLRTIANDCELCVNVACTGCL
`15 FKTLRTIANDDCELCVNVACTGCL
`14 NDDCELCVNVACTGCL
`13 PNTCEICAYAACTGC
`12° PSTCEICAYAACAGC
`I 1'· EDPGTCEIC(Acm)AYAACTGC(AcmJ
`IOh EDPGTC(Acm)EICAYAAC(Acm)TGC
`9 EDPGTCEICVNVACTGC
`8 EDPGTCEICAGGACTGC
`7 EDPGCEICAYAACTGC
`6 EDPGTCICAYAACTGC
`5" EDPGTCEICAYAACTGC
`4 EDPGTCEICAYAACTGC
`3 PGTCEICAYAACTGC
`2 TCEICAYAACTGC
`I CEICAYAACTGC
`
`Amino acid composition
`
`No. Sequence
`
`Analytical data of synthetic peptides
`
`TABLE l
`
`Bausch Health Ireland Exhibit 2020, Page 5 of 9
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`troduction of disulfides did not alter the low-temperature
`HPLC pattern of guanylin-(99-115) (5).
`Rechromatography of the collected peaks I and II
`from an analytical HPLC run of guanylin 4 at 11 °C
`(Fig. 3b) unexpectedly did not result in single peaks.
`Proving the interconvertibility between the two iso(cid:173)
`mers, the mixture of both compounds was recovered
`after HPLC of isolated peaks I and II (Fig. 3d). There(cid:173)
`fore, racemization of cysteine residues during peptide
`assembly can be excluded as the reason for two HPLC
`peaks with identical molecular weights. Although our
`attempts to separate both isoforms failed, repeated
`cycles of analytical HPLC, peak collection and
`rechromatography led to concentrated peaks I and II,
`respectively (Fig. 3d). Variation of the conditions of
`HPLC analysis of peptides 3 and 4, the HPLC (phos(cid:173)
`phate buffer, pH 3) and storage of the collected
`isoforms corresponding to peaks I and II (Fig. 3, a
`and b) for different periods (up to 30 days) at differ(cid:173)
`ent temperatures, even at +50 °C and liquid nitrogen
`temperature, did not change the original HPLC char(cid:173)
`acteristics. From the results of NMR spectroscopy, the
`half-life of guanylin isomers was estimated to be at
`least in the range of seconds (18). Regarding the
`rechromatography of the collected isoforms, which was
`performed about 90 min after collection of both iso(cid:173)
`mers, a maximum half-life of 90 min of the isoforms
`can be concluded. However, the mechanism of
`interconversion remains unclear. Because of a high ac-
`
`GC-C-activating peptides guanylin and uroguanylin
`
`tivation barrier for a mutual exchange of isoforms,
`which has been described to be in excess of 20 kcal/
`mol (18), an intermediate opening of one disulfide loop
`followed by a cystine rearrangement may be consid(cid:173)
`ered as an alternative mechanism for the formation of
`topoisomers. This pathway seems to be unlikely be(cid:173)
`cause the formation of positional disulfide isomers by
`the attack of a free thiol on the second disulfide would
`be a likely side effect. There are no experimental indi(cid:173)
`cations for a cystine rearrangement. Recently, Chino et
`al. (21) reported the isolation of two isoforms of syn(cid:173)
`thetic uroguanylin-(97-112) (14). Depending on the
`order of introduction of the disulfide bonds, the isomers
`were formed in a different ratio. Although we can con(cid:173)
`firm this result, only one compound was isolated after
`synthesis of uroguanylin-(89-112) (15) and other
`uroguanylins (Fig. 3c). Our preliminary NMR studies
`of synthetic peptide 15 demonstrated that only one
`compound is present in solution (data not shown).
`However, it is possible that the second isomer was not
`noticed during chromatographic product separation.
`Our results show that guanylins 3 and 4 exist as a
`mixture of two isoforms which are interconverted. As
`suggested for uroguanylin (21), the order of introduc(cid:173)
`tion of the two disulfide loops had no influence on the
`product composition. This was revealed by synthetic
`peptides 4 and 5 which showed an identical HPLC pat(cid:173)
`tern. An amino-terminal truncation of the first two resi(cid:173)
`dues of guanylin in peptide 2 only influences the
`
`(a)
`
`(b)
`
`35 °C
`
`§
`-N
`"'
`.;
`<J " i::: -e 0
`"'
`.r:, < __ )
`
`(c)
`
`(d)
`
`25 °C
`
`35 °c
`
`11 °C
`
`11 °C
`Peak I'\ /Peak II
`
`11 °C
`
`11 °C
`
`20
`
`.lO
`
`40
`
`20
`
`30
`
`40
`
`40
`
`50
`
`25
`
`30
`
`35
`
`Time (min)
`
`FIGURE3
`(a) HPLC analysis of guanylin-(101-
`115) 3 at 11, 25 and 35 °C; (b) HPLC
`analysis of guanylin-(99-115) 4 at II,
`25 and 35 cc; (c) HPLC analysis of
`uroguanylin-(89-112) 15 at 11 and
`35 cc; (d) HPLC of peak I (bottom)
`and II (top) at 11 cc obtained after
`collection from run (b), peaks were
`separated two times and reanalyzed
`under the same conditions. Gradient:
`15-45% buffer B in 60 min.
`
`227
`
`Bausch Health Ireland Exhibit 2020, Page 6 of 9
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`J. Klodt et al.
`
`detectability of the two isomers by HPLC. Mutations
`or deletions at the N-terminal region of peptides 1, 7,
`9, 12 and 13, and the C-terminal mutation in 12 (Thr
`to Ala) also resulted in two HPLC peaks. In contrast,
`the hybrid mutant 9 appears as a single peak, but con(cid:173)
`sists of two compounds as detected by two distinct sets
`of NMR resonances ( data not shown). Chino et al. (2 I)
`suspected that the carboxy-terminal leucine residue
`present in uroguanylin-(97-112) (14) may stabilize the
`isomers because the absence of this leucine residue re(cid:173)
`sulted in a peptide showing a HPLC characteristic simi(cid:173)
`lar to guanylin. On the other hand, uroguanylin 19 with
`a carboxy-terminal cysteine was homogeneous during
`reverse-phase HPLC. Summarizing the present data, it
`is possible that a C-terminally attached elongation is
`crucial for the stability of topoisomers, but other resi(cid:173)
`dues, in particular between the inner cysteine residues,
`also influence this phenomenon by their sterical bulk.
`Deduced theoretically by Mao (34 ), guanylin and
`uroguanylin represent an actual example of topologi(cid:173)
`cal stereoisomerism. Further studies dealing with the
`sequence dependency of the formation of guanylin
`isoforms and of the influence of variables like pH, sol(cid:173)
`vent and temperature are currently under investigation
`in our laboratory. Stability and NMR studies of syn(cid:173)
`thetic uroguanylins 14 and 15 are also in progress.
`
`Biological activity
`Guanylin and uroguanylin peptides had a concentra(cid:173)
`tion-dependent effect on the intracellular cGMP level
`at concentrations between 1 o-8 and I o---o M, whereas E.
`coli enterotoxin STa was already active at 10-9 M (Fig.
`4a). Different monocyclic derivatives, isolated as in(cid:173)
`termediates during synthesis, were biologically inac(cid:173)
`tive indicating that the two disulfide bonds and,
`therefore, the rigid three-dimensional structure is nec(cid:173)
`essary for receptor activation. Guanylin lacking
`Thrl03, either as a truncated (1) or deletion peptide
`(7), was only effective at a concentration of 10-6 :-.1.
`Thus, Thr103 is a crucial residue for receptor activa(cid:173)
`tion. This finding corresponds with earlier reports about
`the ability of rat guanylin to bind to intestinal mem(cid:173)
`branes (35). Interestingly, amino acid exchange of resi(cid:173)
`due 102 of guanylin did not influence its activity on
`cGMP generation (Fig. 4a, compounds 12 and 13). Pep(cid:173)
`tide 6 lacking the endocyclic GlulOS was inactive. Re(cid:173)
`versing the order of disulfide introduction in human
`guanylin-(99-115) had no effect on HPLC pattern and
`did not influence its bioactivity (4 and 5). From this re(cid:173)
`sult, it can be concluded that peptides 4 and 5 have a
`similar composition. Compound 8 with the non(cid:173)
`voluminous residues AGGA between the inner cys(cid:173)
`teines was inactive, indicating an important role for this
`region in receptor activation. For uroguanylin peptides,
`changes in the N-terminal region, like a specific
`biotinylaminocaproylation (20) or amino acid exchange
`(19) did not influence the increase of cGMP concen-
`
`228
`
`rA)
`
`Bil i0-9M ■ 10-8M
`
`□ 111-JM O I0-6M
`
`I
`
`2
`
`3
`
`.1
`
`'.'i
`
`6
`
`I
`
`8
`
`9
`
`10
`
`II
`
`12
`
`11 ST:t C
`
`/
`
`/
`
`/
`
`Peptide
`
`Gl !0-9\1 ■ ID-8\-f
`
`J
`l , ,.
`;, \,#',II~ I
`
`!8
`
`l9
`
`20
`
`STa
`
`!-1
`
`!5
`
`16
`
`l7
`
`Peptide
`
`FIGURE 4
`Effects of synthetic peptides on intracellular cGMP concentration
`of cultured T84 cells. (a) Guanylin-related peptides and (b)
`uroguanylin derivatives (C = control; approximately 106 cells/
`well; 50 µL/well).
`
`tration in T84 cells (Fig. 4b ). It is noticeable that dele(cid:173)
`tion of Glu 10 I (17) did not influence the bioactivity of
`uroguanylin, whereas deletion of Asp99 resulted in a
`peptide (16) with only minor activity. Superficially, the
`latter effect seems to be similar to guanylin 7 lacking
`residue Thrl03, but the second Asp residue of
`uroguanylin complicates interpretation of a functional
`connection between reduction in bioactivity and the
`amino acid residue 103 in guanylin and residue 99 in
`uroguanylin. An unexpected result was obtained from
`the cGMP bioassay of compound 9. Being a hybrid of
`guanylin and uroguanylin, this compound had only mi(cid:173)
`nor activity at 10-7 M.
`In the intestinal epithelium, a final effect of GC-C(cid:173)
`activating peptides is the cGMP-mediated activation of
`the CFTR (cystic fibrosis transmembrane conductance
`regulator) chloride channels, thereby enhancing chlo(cid:173)
`ride secretion (1, 2, 5). Selected synthetic peptides were
`tested in their ability to modulate ion transport using
`rat intestinal mu