The Role of the Merrifield Solid Phase Method in the Discovery and Exploration of a New Class of Selective Vasopressin Hypotensive Agonists

The Role of the Merrifield Solid Phase Method in the Discovery

and Exploration of a New Class of Selective VasopressinHypotensive Agonists*

Maurice Manning,1,3

Stoytcho Stoev,1Ling Ling Cheng,

1Nga Wo,

2W. Y. Chan,

2and

Hazel H. Szeto2

(Accepted January 28, 2007; Online publication March 10, 2007)

The Merrifield solid phase method (MSPM) has been of inestimable value in studies aimed at

the design of selective agonists and antagonists of the V1a (vascular), V1b (pituitary), V2 (renal)and OT (uterine) receptors for vasopressin (VP) and oxytocin (OT). Here we describe how,40 years after the landmark syntheses of oxytocin and vasopressin by Vincent du Vigneaudand colleagues, it led to the discovery of a new class of vasopressin agonists which exhibit

selective hypotensive activity. We also point to the role of serendipity in this discovery. Wefurthermore show how the MSPM has been of inestimable value in facilitating a rapid andcomprehensive structure activity study of the lead hypotensive peptide: d(CH2)5[D-

Tyr(Et)2,Arg3,Val4]AVP (A) giving rise to d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Lys7,Eda9]LVP(B) which exhibits a 30-fold enhancement in vasodepressor potency relative to (A). Here, wealso report a structure activity study of (B) with single modifications at position 3 (Lys, Nar)

and 4 (Cha, Nle, Leu, Abu, Nva, Thr, Har) and combined modifications at positions 3 and 9(Nar and EdaG) and 7 and 9 (Arg and Eda ‹ retro Tyr). All modifications of (B) are welltolerated with good retention of vasodepressor potency. These findings offer promising cluesto the design of more potent VP hypotensive agonists and of critically needed antagonists of

the putative VP vasodilating receptor. The Merrifield Solid Phase Method will continue toplay a pivotal role in these studies.

KEY WORDS: solid phase synthesis; vasopressin; hypotensive peptides.

Abbreviations: Symbols and abbreviations are in accordance with

the recommendations of the IUPAC-IUB Commission on Bio-

chemical Nomenclature (Eur. J. Biochem. 1989, 180, A9–A11) and

IUPHAR (Trends Pharmacol. Sci. (2001), and as follows. All

amino acids are in the L-configuration unless otherwise noted.

Other abbreviations used are:AVP, arginine vasopressin; LVP,

lysine vasopressin; VP, vasopressin; OT, oxytocin, D-Tyr(Et),

O-ethyl-D-tyrosine; D-Tyr(Pri), O-i-propyl-D-tyrosine; Abu,

2-aminobutanoic acid; Aic, 2-aminoindane-2-carboxylic acid; Atc,

2-aminotetraline-2-carboxylic acid; Cha, 1-amino-cyclopentane-1-

carboxylic acid (cyclohexylalanine); Oic, Octahydroindole-2-car-

boxylic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

* Dedicated to the memory of Professor Bruce Merrifield.1 Department of Biochemistry and Cancer Biology, The University

of Toledo, Mail Stop #1010, Health Science Campus, 3000

Arlington Avenue, Toledo, OH, 43614-2598, USA.2 Department of Pharmacology, Weill Medical College of Cornell

University, New York, NY, 10021, USA.3 Correspondence should be addressed to: Maurice Manning,

Department of Biochemistry and Cancer Biology, The University

of Toledo, Mail Stop #1010, Health Science Campus, 3000

Arlington Avenue, Toledo, OH, 43614-2598, USA. Tel.: +1-419-

3834131; Fax: +1-419-3836228; e-mail: maurice.manning@

utoledo.edu

International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 1–2, June 2007 (� 2007) pp. 7–17

DOI: 10.1007/s10989-007-9089-9

71573-3149/07/0600–0007/0 � 2007 Springer Science+Business Media, LLC

INTRODUCTION

Bruce Merrifield introduced his now famous SolidPhase Method for the synthesis of peptides in 1963(Merrifield et al., 1963). This publication has sincebecome the 5thmost highly cited paper in the history ofthe Journal of the American Chemical Society (Rawls,2003; Stang, 2003). In 1984, Bruce Merrifield wasawarded theNobel Prize inChemistry in recognition ofthe widespread impact of this method not only onpeptide chemistry but on the entire field of syntheticorganic chemistry. The solid phase synthesis of oxy-tocin and of deamino oxytocin were reported in 1968(Manning, 1968; Takashima et al., 1968). Since then,the solid phase method has proved to be of inestimablevalue for the synthesis of selective agonists andantagonists of oxytocin and of vasopressin (for reviewssee: Hruby and Smith, 1987; Lebl, 1987; Lebl et al.,1987; Manning and Sawyer, 1989, 1993; Manninget al., 1995). In addition to its well known renal anti-diuretic effects, mediated by the V2 receptor, its vaso-pressor effects,mediated by theV1a receptor, itsACTHreleasing effects mediated by the V1b receptor and itsuterine contracting effects, mediated by the oxytocin(OT) receptor (for reviews see: Jard, 1998; Barberiset al., 1999) vasopressin also causes vasodilation(Katusic et al., 1984; Liard, 1989; Hirsch et al., 1989).However, the receptor whichmediates this effect has todate not been characterized. Within the past decade,we discovered a new class of vasopressin agonistswhich exhibit selective vasodepressor i.e. hypotensiveactivity (Chan et al., 1998a, b; Manning et al., 1998).These are the first such agonists for the putative VPvasodilating receptor. Here, we will recount how thecombination of the Merrifield solid phase method, rat

bioassays and serendipity led to this discovery and howthe Merrifield solid phase method has greatly facili-tated structure activity studies on this new class of VPagonists (Manning et al., 1999a, b, c; Chan et al., 2000,2001a; Cheng et al., 2001; Stoev et al., 2004, 2005).

How the Merrifield Method Facillated this Discovery

The synthesis of oxytocin (OT) and of argininevasopressin (AVP) by du Vigneaud and colleagues byclassical methods of peptide chemistry (du Vigneaudet al., 1954a, b) are true milestones in the history ofpeptide science. These syntheses led to intense interestin structure activity studies on OT and VP (Berde andBoissonnas, 1968). Utilization of the Merrifield solidphase method for the synthesis of oxytocin (Man-ning, 1968) and deamino oxytocin (Takashima et al.,1968) had a truly catalytic impact on structureactivity and design studies on oxytocin and vaso-pressin (Hruby and Smith, 1987; Lebl, 1987; Leblet al., 1987; Manning and Sawyer, 1989, 1993;Manning et al., 1995). Use of the Merrifield solidphase method brought about a 20–30-fold increase inthe number of analogs that could be synthesizedusing classical methods of peptide synthesis. Thus inthe du Vigneaud laboratory, the synthesis of oneanalog of oxytocin using solution methods could takeanywhere from 6 months to 1 year. With the solidphase method, the synthesis of 25–30 analogs/year bya single individual soon became the standard. Thiscan also be readily observed by checking the differ-ences in the number of analogs reported in a publi-cation which utilized classical methods of peptidechemistry (Manning et al., 1965) with those in morerecent publications which utilizes the solid phasemethod (Cheng et al., 2004). This capability meantthat (a) leads could be followed up more rapidly and(b) entire series of analogs could be synthesized andevaluated in pharmacological assays. These advan-tages proved to be crucial to the discovery of a novelclass of vasopressin agonists (Chan et al., 1998a, b;Manning et al., 1998), 40 years after the historicsyntheses in the du Vigneaud laboratory of oxytocin(du Vigneaud et al., 1954a) and arginine vasopressin(du Vigneaud et al., 1954b).

Discovery of Novel Vasopressin Hypotensive Agonists

Remarkably this discovery emanated from anintensive study of vasopressin V2/V1a/OT antago-nists. In collaborative studies with Wilbur H. Sawyer,Serge Jard and Claude Barberis we had utilized the

(OH)Tic, 1,2,3,4-tetrahydroisoquinoline-7-hydroxy-3-carboxylic

acid; Har, homoarginine, Nar, norarginine; Nva, norvaline; Eda,

ethylenediamine; EdaG, 1-amino-2-guanidinoethane; d(CH2)5,

b-mercapto-b,b-pentamethylenepropionyl; d(CH2)5(Mob), b-(4-methoxybenzyl)mercapto-b,b-pentamethylenepropionyl; Eda ‹ Tyr,

Eda retro-tyrosine; d(CH2)5[D-Tyr(Et)2]VAVP, [1-(b-mercapto-

b,b-pentamethylene propionic acid) -2-O-ethyl-D-tyrosine, 4-

valine]arginine vasopressin; d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP

(A), [1-(b-mercapto-b,b-pentamethylene propionic acid) -2-O-

ethyl-D-tyrosine, 3-arginine, 4-valine]arginine vasopressin; DMF,

dimethylformamide; DCC, dicyclohexylcarbodiimide; HOBt, 1-

hydroxybenzotriazole, ONp, 4-nitrophenyl ester; Boc, t-butyloxy-

carbonyl; Bzl, benzyl; Tos, tosyl; Z, benzyloxycarbonyl; Mob, 4-

methoxybenzyl; Z(2Cl), 2-chlorobenzyloxycarbonyl; AcOH, acetic

acid; TFA, trifluoroacetic acid; DIPEA, diisopropylethylamine;

TLC, thin layer chromatography; HPLC, high performance liquid

chromatography, ESMS, electro-spray mass spectrometry; AUC,

area under the vasodepressor response curve; ED, effective dose.

8 Manning et al.

Merrifield solid phase method to synthesize a widevariety of non-selective and selective cyclic, linear andradioiodinatable V1a, V2 and OT antagonists (forreviews see: Manning and Sawyer, 1989, 1993;Manning et al., 1995). The first VP hypotensiveagonist was unexpectedly discovered through acombination of persistence and luck while takingadvantage of the opportunity offered by the Merri-field solid phase method to synthesize a large analogseries followed by a thorough examination of theirpharmacological properties in rat bioassays. We hadused this approach to very good effect in the design ofthe first selective antagonist of the rat V2 (antidi-uretic) receptor, d(CH2)5[D-Ile2,Ile4]AVP (Manninget al., 1984). Briefly, we had taken as a lead the non-selective V2/V1a/OT antagonist d(CH2)5[D-Tyr(-Et)2,Val4] AVP (Manning et al., 1982) which has thefollowing structure:

d(CH2)[D-Tyr(Et)2,Val4]AVP was modified at posi-tions 2 (with aliphatic and aromatic D-amino acids)and 4 (with aliphatic amino acids). This strategy led tothe discovery of d(CH2)5[D-Ile2,Ile4]AVP (Manninget al., 1984). Much later, we embarked on a study ofposition 3 in d(CH2)5[D-Tyr(Et)2,Val4]AVP (Manninget al., 1997). Incidentally, during the almost 40 yearssince the syntheses of VP and OT, position 3 had beenvirtually ignored in structure/activity studies of thesepeptides: We replaced the Phe3 residue in d(CH2)5[D-Tyr(Et)2,Val4]AVP with 15 different conformationallyrestricted, aromatic, aliphatic and basic amino acids.Remarkably, the conformationally restricted aminoacids, Tic, (HO)Tic, Pro, Oic, Atc, D-Atc, Aic, thearomatic amino acids, Tyr, Trp, Hphe and the ali-phatic amino acids Ile, Leu are very well tolerated withmoderate to full retention of antagonism for one ormore of the V1a, V2 and OT receptors (Manning et al.,1997). From these findings, it appeared that position 3in d(CH2)5[D-Tyr(Et)2,Val4]AVP was fully tolerant ofstructural change. Imagine our surprisewhenwe foundthat the replacement of the Phe3 residue byArg3 to gived(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP (A) not only to-tally abolished the V1a, V2 and OT antagonism of theparent peptide but manifested totally unexpectedhypotensive agonism (Chan et al., 1998a, b).

Thus, d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP (A)which has the following structure:

is the first reported analog of VP or OT to exhibitselective vasodepressor activity (Chan et al., 1998b).It offered evidence that the well-known vasodilatoryeffect of VP (Liard 1989; Katusic et al., 1984; Hirschet al., 1989) could be mediated by a hitherto un-characterized receptor. This unexpected findingopened up the possibility of designing more potentagonists, antagonists and radioiodinated ligands forthis putative VP vasodilating receptor.

The Role of Serendipity in the Discovery of the Novel

Selective VP Hypotensive Peptides

From the forgoing, it can be clearly seen that theMerrifield solid phasemethod, in combinationwith thestandard rat bioassays, played a huge role in the dis-covery of the first novel selective VP hypotensivepeptides. Sowhere and howdid serendipity play a role?The role of luck did not become apparent until webegan to examine in rat bioassays and particularly inthe rat vasodepressor assay the effects of an Arg3/Phe3

interchange in three other VP antagonists. These wereNo. 1: the selective AVP V1a /OT antagonistd(CH2)5[Tyr(Me)2]AVP, (Kruszynski et al., 1980) No.2: the selective AVP V2 antagonist d(CH2)5[D-Ile2,I-le4,Ala-NH2

9]AVP (Sawyer et al., 1988) and No. 3: thelinear V2/V1a antagonist: Aaa-D-Tyr(Et)2,Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (Manning et al., 1987a,b). To our surprise, replacement of the Phe3 residue inthese three peptides by anArg3 residue did not result inpeptides which exhibited hypotensive activity(Manning et al., 1999a). Thus had we selected any oneof these three peptides instead of d(CH2)5[D-Tyr(-Et)2,Val4]AVP for a structure/activity study of posi-tion 3, the discovery of this new class of hypotensiveagonists would not have been made. So clearly theselection of the V2/V1a antagonist, d(CH2)5[D-Tyr(-Et)2,Val4]AVP (Manning et al., 1982) for the study ofposition 3 changes which led to the discovery ofd(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP, the first AVPhypotensive agonist, was a most fortuitous choice.

The Merrifield Solid Phase Method Greatly

Facilitated the Rapid Structure Activity Investigation

of the Vasopressin Hypotensive Agonist d(CH2)5[D-

Tyr(Et)2,Arg3,Val4]AVP (A)

Hypotensive potencies are measured as thevasodepressor effective dose (ED) in lg/100 g i.v. The

1 2 3 4 5 6 7 8 9 CH 2-CO-D-Tyr(Et)-Phe-Val-Asn-Cy-Pro-Arg-Gly-NH2

S S

1 2 3 4 5 6 7 8 9 CH2-CO-D-Tyr(Et)-Arg-Val-Asn-Cy-Pro-Arg-Gly-NH2 (A)

S S

Solid Phase Method Leads to Discovery of Vasopressin Hypotensive Agonists 9

ED is the dose that produces a vasodepressor re-sponse of 5 cm2 AUC (area under the curve) in the 5-min period following injection of the test peptide (seesection on Bioassays for how EDs are measured).Peptide (A) exhibits a vasodepressor ED = 4.66 lg/100 g (Chan et al., 2001a). This is the standardagainst which all new VP hypotensive peptides ana-logues are measured. Peptide A became the leadcompound in extensive structure/activity relationshipstudies aimed at delineating which structural featuresof (A) are required for hypotensive activity (Manninget al., 1999a, b, c; Chan et al., 2000, 2001; Chenget al., 2001, 2006; Stoev et al., 2005, 2006). Thesestudies, which required the solid phase synthesis of awide variety of analogs of (A), have shown that therequirements for hypotensive activity are quite rigid(Manning et al., 1999a, b, c; Chan et al., 2000, 2001).However, we also found that (A) could be modified togive peptides with enhanced hypotensive potencies(Manning et al., 1999a, b, c; Chan et al., 2000, 2001;Cheng et al., 2001, 2006). We recently reported (Stoevet al., 2006) that the D-Tyr(iPr)2, Lys7, Lys8, Eda

9

(ethylenediamine) analog of (A), d(CH2)5D-Tyr(-iPr)2,Arg3,Val4,Lys7,Eda9]LVP (B) exhibits anED = 0.15 lg/100g; a 30-fold increase in hypoten-sive activity relative to (A) (Stoev et al., 2006). (B) hasthe following structure:

Using (B) as a new lead, we now wish to report astructure activity study of (B) with single modificationat positions 3 and 4 and with combined modification atpositions 3 and 9 and at positions 7 and 9. The position3 substitutions are: Lys and Nar: The position 4 sub-stitutions are: Abu, Nva, Leu, Nle, Cha, Thr and Har.The positions 3 and 9 substitutions are: Nar and EdaG.The positions 7 and 9 substitutions are: Arg and Edaretro Tyr. The resulting 11 new peptides have the fol-lowing general structure:

METHODS

Bioassays

Peptides were assayed for agonistic activity orantagonistic activity in the rat antidiuretic assay,vasopressor assay and in vitro rat oxytocic assay. Foragonists, the 4-point assay design was used (Holton,1948) and for antagonists, Schild�s pA2 method wasemployed (Schild, 1958). In the rat in vivo assays, theeffective dose (ED) of antagonist is divided by anarbitrarily assumed volume of distribution of 67 ml/kg to allow estimation of its molar concentration forpA2. Synthetic arginine-vasopressin and oxytocinwhich had been standardized in vasopressor andoxytocic units against the UPS Posterior PituitaryReference Standard were used as working standardsin all bioassays. Antidiuretic assays were on water-loaded rats under ethanol anesthesia as described in(Sawyer, 1961). Vasopressor assays were performedon urethane-anesthetized and phenoxybenzamine-treated rats as described by (Dekanski, 1952). Oxy-tocic assays were performed on isolated uteri fromdiethylstilbestrol-primed rats in a Mg2+-free VanDyke-Hasting�s solution (Munsick, 1960).

Vasodepressor Assays

The vasodepressor activity of the hypotensivepeptides was determined in urethane-anesthetizedmale rats (weight 200 to 250 g) as previouslydescribed (Chan et al., 2001a). Blood pressure (BP)was monitored via a cannulated carotid artery. Sincethe vasodepressor response is baseline BP dependent

Peptide X3

Y4

Z7

W9

B Arg Val Lys Eda1 Lys Val Lys Eda2 Nar Val Lys Eda3 Arg Cha Lys Eda4 Arg Nle Lys Eda5 Arg Leu Lys Eda6 Arg Abu Lys Eda7 Arg Nva Lys Eda8 Arg Thr Lys Eda9 Arg Har Lys Eda10 Nar Val Lys EdaG11 Arg Val Arg Eda ‹ Tyr

A preliminary report on these peptides was recently presented(Cheng et al., 2006)

1 2 3 4 5 6 7 8 9 CH2-CO-D-Tyr(iPr1)-Arg-Val-Asn-Cy-Lys-Lys-Eda (B)

S S

1 2 3 4 5 6 7 8 9 CH2-CO-D-Tyr(Pri) X-Y-Asn-Cy-Z-Lys-W

S S

10 Manning et al.

(Chan et al., 2001), rats used for the quantitativebioassays of vasodepressor potencies were given aninfusion of phenylephrine to elevate and maintaintheir baseline BP at 110–120 mm Hg. Phenylephrine(25 lg mL-1) was infused at a rate(0.01–0.05 mL min)1) to maintain the baseline BP atthe required range for the 5-minute period before theinjection of the test peptide. The infusion was con-tinued for another 5-minute period following thepeptide injection and then ceased. Upon recovery ofthe vasodepressor response, phenylephrine infusionwas reinstituted for the next peptide injection. Two tothree peptide injections could be administered in astable preparation.

The vasodepressor potency was measured by thearea under the vasodepressor response curve (AUC),determined by a polar planimeter, for the 5-minuteperiod following the injection of the hypotensive VPpeptide. Two predetermined doses of the test peptidewere injected: a low dose that would produce avasodepressor AUC response of less than 5 cm2

during the 5-minute period following the peptideinjection and a high dose that would produce anAUC response of greater than 5 cm2. Four to six ratswere used for each peptide to obtain the meanthreshold dose. The mean responses to the low andthe high doses were computed and the two pointdose-response curve constructed. The dose thatwould produce an AUC response of 5 cm2 wasinterpolated from the two point dose-response curve.This calculated dose, in lg 100)1 g, (of rat used in theexperiment) is the effective dose (ED) for the 5 cm2

AUC response and was used to express the vasode-pressor potency of the hypotensive peptide.

Peptide Synthesis

Peptides 1–11 (Tables I and II) were synthesizedutilizing the Merrifield solid phase method or by acombination of solid-phase and solution synthesismethods. For the preparation of the protected pep-tide II, first, d(CH2)5(Mob)-D-Tyr(Pri)-Dap(Fmoc)-Val-Asn-Cys(Mob)-Lys[Z(2Cl)]-Lys[Z(2Cl)]-resin wasprepared, followed by side chain Fmoc group re-moval and transformation of the Dap3 residue intothe Nar3 residue by guanidilation on the resin using1-H-Pyrazole-1-carboxamidine hydrochloride/DI-PEA (Bernatowicz et al., 1992). An aliquot of theprotected precursor of peptide II was subjected toguanidinylation in solution by treatment with 1-H-Pyrazole-1-carboxamidine hydrochloride/DIPEA inDMF at 48–50�C for 6 h (Bernatowicz et al., 1992) togive the protected peptide (No. X, Table III). Theprotected retroTyr peptide (No. XI, Table III) wasprepared by a combination of solid-phase (Merrifield,1964) and solution methods. First, the solid-phasemethod was utilized to prepare the appropriateintermediate protected Eda acylheptapeptide, fol-lowed by a DCC/HOBt-mediated (Konig and Geiger,1970) ‘‘7 + 1’’ coupling in solution with Z-Tyr(Bzl)as described in (Manning et al., 1992). Solid-phasesyntheses were carried out by the Merrifield method(Merrifield, 1964) with the modifications previouslydescribed (Kruszynski et al., 1980; Manning, 1968;

Table I. Vasodepressor Potencies of analogues of d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Lys7,Eda9]LVP (B) with single (positions 3 and 4) andcombined modifications (positions 3 and 9 and positions 7 and 9)

No Peptide Vasodepressor ED, lg/100 ga,b

A d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP(A)c 4.66±0.46B d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Lys7,Eda9]LVPd 0.15±0.011 d(CH2)5[D-Tyr(Pri)2,Lys3,Val4,Lys7,Eda9]LVPe 0.23±0.052 d(CH2)5[D-Tyr(Pri)2,Nar3,Val4,Lys7,Eda9]LVPe 0.30±0.013 d(CH2)5[D-Tyr(Pri)2,Arg3,Cha4,Lys7,Eda9]LVPe 0.22±0.034 d(CH2)5[D-Tyr(Pri)2,Arg3,Nle4,Lys7,Eda9]LVPe 0.36±0.015 d(CH2)5[D-Tyr(Pri)2,Arg3,Leu4,Lys7,Eda9]LVPe 0.45±0.056 d(CH2)5[D-Tyr(Pri)2,Arg3,Abu4,Lys7,Eda9]LVPe 0.54±0.057 d(CH2)5[D-Tyr(Pri)2,Arg3,Nva4,Lys7,Eda9]LVPe 0.79±0.038 d(CH2)5[D-Tyr(Pri)2,Arg3,Thr4,Lys7,Eda9]LVPe 0.78±0.059 d(CH2)5[D-Tyr(Pri)2,Arg3,Har4,Lys7,Eda9]LVPe 1.92±0.210 d(CH2)5[D-Tyr(Pri)2,Nar3,Val4,Lys7,EdaG9]LVPe 0.21±0.017C d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Lys7,Eda ‹ Tyr10]LVPd 0.14±0.0211 d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Arg7,Eda ‹ Tyr10]LVPe 0.25±0.02

aED, effective dose (in lg 100g)1i.v.) is the dose that produces a vasodepressor response of 5cm2 AUC in the 5-min period following injectionof test peptide. AUC, area under the vasodepressor response curve (see section on Methods for details of vasodepressor assays).ball peptides exhibited undetectable or negligible agonistic or antagonistic activities in the standard antidiuretic, vasopressor and oxytocin(in vitro, no Mg2+) assays.cData from ref. (Chan et al., 2001a). dData from ref. (Stoev et al., 2006). eThis publication.


Manning et al., 1982, 1999b). The acylpeptide resinswere cleaved using aminolysis with ethylenediamine(Eda) in methanol (Glass and du Vigneaud, 1973,Manning et al., 1992) to give the protected Edapeptides I-IX (Table III). The physiocochemicalproperties of all protected peptides are given inTable III. Deprotection of all protected peptides wascarried out with sodium in liquid ammonia (duVigneaud et al., 1954a, b). The disulfide formationand purification procedures to give the free peptides1–11 (Tables I and II) were performed as previouslydescribed (Manning et al., 1999b).

RESULTS AND DISCUSSION

Peptides 1–11 (see structures above) were assayedfor agonism and antagonism in VP vasopressor,antidiuretic, oxytocic (in vitro) and VP vasodepressorassays as described above. All peptides exhibit neg-ligible agonistic or antagonistic, antidiuretic, vaso-pressor and oxytocic activities. Their vasodepressorpotencies are presented in Table I. All new peptidesalso exhibit significant enhancement of vasodepressorpotency relative to the original hypotensive peptide(A) (Table I).

Effects of Positions 3 Modifications of (B) (Peptides 1

and 2)

We replaced the Arg3 residue in (B) with Lys andNar residues to give peptides Nos. 1 and 2 (Table I).With ED values of 0.23 lg/100 g and 0.30 lg/100 g

respectively, peptides 1 and 2 are 15 and 20 times,respectively more potent than peptide (A) and slightlyless potent than peptide (B).

Effects of Position 4 Modifications of (B)

(Peptides 3–9)

Position 4 in (B) was modified by replacement ofthe Val4 residue with a wide range of aliphatic (Cha,Nle, Leu, Abu, and Nva), polar (Thr) and charged(Har) amino acid residues to give peptides Nos. 3–9(Table I). All position 4 substitutions in (B) are well-tolerated. With an ED = 0.22 lg/100 g, the Cha4

analog (No. 3) is the most potent of this series.Peptides 3–9 are 2.4 to 21 times more potent thanpeptide (A). They all however exhibit somewhatlower vasodepressor potencies than the parent pep-tide (B).

Effects of Combined Modifications of (B) at Positions

3 and 9 and Positions 7 and 9 (Peptides 10 and 11)

Positions 3 and 9 of peptide (B) were modified byArg3/Nar3 and Eda9/1-amino-2-guanidinoethane(EdaG)9 interchanges to give peptide No. 10 (Ta-ble I). With an ED = 0.21 lg/100 g, peptide No. 10is 1.5 times more potent than its parent peptide No. 2(Table I), but is still slightly less potent than peptide(B). Modification of peptide (B) at positions 7 and 9by replacement of Lys7 and Eda9 with Arg7 andEda ‹ Tyr9 respectively, resulted in the radioiodin-atable peptide No. 11 (Table I). Peptide 11 is the Arg7

analogue of the previously reported radioiodinatable

Table II. Physicochemical properties of free-peptides, 1 –11a

No Peptide Yield(%)b TLC, Rfc HPLC tR

(min)Formula MW MW

Founda c d f

1 d(CH2)5[D-Tyr(Pri)2,Lys3,Val4,Lys7,Eda9]LVP 14.5 0.16 – 0.28 0.05 16.8 C52H89O10N13S2 1120.4 1119.42 d(CH2)5[D-Tyr(Pri)2,Nar3,Val4,Lys7, Eda9]LVP 15.9 0.19 – 0.32 0.06 18.2 C51H87O10N15S2 1134.5 1135.23 d(CH2)5[D-Tyr(Pri)2,Arg3,Cha4,Lys7, Eda9]LVP 32.2 0.18 – 0.31 0.11 19.8 C56H94O10N15S2 1202.5 1202.74 d(CH2)5[D-Tyr(Pri)2,Arg3,Nle4,Lys7, Eda9]LVP 19.5 0.15 0.03 0.34 0.06 17.2 C53H91O10N15S2 1162.6 1162.35 d(CH2)5[D-Tyr(Pri)2,Arg3,Leu4,Lys7, Eda9]LVP 36.2 0.15 0.03 0.34 0.07 17.0 C53H91O10N15S2 1162.6 1162.66 d(CH2)5[D-Tyr(Pri)2,Arg3,Abu4,Lys7, Eda9]LVP 24.6 0.15 – 0.28 0.03 17.1 C51H87O10N15S2 1134.4 1133.87 d(CH2)5[D-Tyr(Pri)2,Arg3,Nva4,Lys7, Eda9]LVP 31.6 0.18 – 0.29 0.08 16.6 C52H89O10N15S2 1148.4 1148.28 d(CH2)5[D-Tyr(Pri)2,Arg3,Thr4,Lys7, Eda9]LVP 40.3 0.11 0.03 0.27 0.04 16.5 C51 H87O11N15S2 1150.5 1150.69 d(CH2)5[D-Tyr(Pri)2,Arg3,Har4,Lys7, Eda9]LVP 23.2 0.06 – 0.28 0.04 15.4 C54H94O10N18S2 1219.5 1219.210 d(CH2)5[D-Tyr(Pri)2,Nar3,Val4,Lys7, EdaG9]LVP 30.2 0.18 – – 0.25 18.9 C52H89O10N17S2 1176.5 1176.911 d(CH2)5[D-Tyr(Pri)2,Arg3,Val4,Arg7,Eda9 ‹ Tyr10]LVP 29.1 0.14 0.05 0.30 0.10 17.8 C61H98O12N18S2 1339.7 1339.9

aPeptides 1–11 were obtained from the corresponding protected peptides I-XI Table III. Yields are based on the amount of protected peptideused in the reduction-reoxidation step in each case and are uncorrected for acetic acid and water content.bSolvent systems and conditions are given in the Experimental.cAll peptides were at least 95% pure. For elution a linear gradient 90:10 to 30:70 (0.05% aqueous TFA: 0.05 TFA in CH3CN ) over 30 minwith flow rate 1.0 ml/min was applied.

12 Manning et al.

peptide (C) (Stoev et al., 2006). Comparison of thevasodepressor potencies of (C) and peptide 11confirm that a Lys7 substitution is superior to anArg7 substitution in leading to enhanced hypotensiveagonism.

CONCLUSION

The Merrifield solid phase method has had anenormous well-documented impact on peptidechemistry in general, on organic chemistry, (Rawlset al., 2003; Stang, 2003) and on the design ofagonists and antagonists for the neurohypophysealpeptides oxytocin and vasopressin (Hruby and Smith,1987; Lebl, 1987; Lebl et al., 1987; Manning andSawyer, 1989, 1993; Manning et al., 1995). In thistribute to dear friend Bruce Merrifield, we recall howthe Merrifield solid phase method, in combinationwith serendipity led to the discovery with rat bioas-says of the first hypotensive vasopressin agonist,d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP (A) (Chan et al.,1998a, b; Manning et al., 1998) and how theMerrifield solid phase method played an invaluablerole in the subsequent structure/activity studies on(A) (Manning et al., 1999a, b, c; Chan et al., 2000,2001; Cheng et al., 2001, 2006; Stoev et al., 2006).These studies led very recently to the newhighly potent hypotensive peptide: d(CH2)5[D-Tyr(-Pri)2,Arg3,Val4,Lys7,Eda9]LVP (B) (Stoev et al.,2006). Furthermore, we report here a structure-activity relationship investigation on the effects ofsingle modifications at positions 3 and 4, and com-bined modifications at positions 3 and 9, 7 and 9 of(B). These studies show that all changes in (B) arewell tolerated, leading to peptides, which like theparent peptide (B), exhibit significant enhancement inhypotensive potencies relative to (A). These newfindings offer promising clues to the design of morepotent VP hypotensive agonists, and of criticallyneeded antagonists of the VP hypotensive response aswell as to the design of radioidinatable and fluores-cent ligands as probes of the putative VP vasodilatingreceptor. Besides being of value as new research toolsfor studies on the multifaceted roles of VP in theregulation of cardiovascular physiology and patho-physiology (Yu et al., 2003; Tabrizchi and Ford,2004), these findings may be of benefit for thedevelopment of a novel class of antihypertensiveagents for therapeutic use. From the forgoing, there islittle doubt that the discovery and development ofthis novel class of vasopressin hypotensive agonists is

Table

III.

Physicochem

icalproperties

ofprotected

peptides

I–XIa

No

Peptide

Yield

(%)b

m.p.(�C)

TLC,R

fc

ab

Cd

ef

Id(C

H2) 5(M

ob)-D-Tyr(Pri)-Lys[Z(2Cl)]-Val-Asn-C

ys(Mob)-Lys[(Z(2Cl)]-Lys[(Z(2Cl)]-Eda

90.0

211–213

0.77

0.62

0.70

0.78

0.98

–II

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Nar-Val-Asn-C

ys(Mob)-Lys[Z(2Cl)]-Lys[Z(2Cl)]-Eda

73.1

176–178

0.47

–0.41

0.67

0.91

0.63

III

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-C

ha-A

sn-C


98.0

192–194

0.75

0.68

–0.89

–0.87

IVd(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-N

le-A

sn-C


89.3

215–217

0.78

0.64

0.69

0.81

0.96

–V

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-Leu-A

sn-C


84.6

205–207

0.75

0.63

0.72

0.80

0.96

–VI

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-A

bu-A

sn-C


97.0

196–198

0.71

0.59

0.70

0.71

0.98

–VII

d(C

H2) 5(M

ob)-D

-Tyr(Pri)-Arg(Tos)-N

va-A

sn-C

ys(Mob)-Lys(

Z(2Cl))-Lys(Z(2Cl)-Eda

91.5

199–201

0.75

0.61

0.70.

0.74

0.98

–VIII

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-Thr(Bzl)-Asn-C


87.8

212–214

0.80

–0.68

0.85

–0.77

IXd(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-H

ar(Tos)-A

sn-C


98.1

193–195

0.75

0.65

–0.87

–0.86

Xd(C

H2) 5(M

ob)-D-Tyr(Pri)-Nar-Val-Asn-C

ys(Mob)-Lys[Z(2Cl)]-Lys[Z(2Cl)]-EdaG

76.9

188–190

0.50

––

0.55

0.92

0.42

XI

d(C

H2) 5(M

ob)-D-Tyr(Pri)-Arg(Tos)-V

al-Asn-C

ys(Mob)-Arg(Tos)-Lys[Z(2Cl)]-Eda

‹Tyr(Bzl)-Z

94.5

207–209

0.86

–0.78

0.88

–0.72

aTheprotected

peptides

I-XIare

theim

mediate

protected

precursors

forthefree

peptides

1–11given

inTablesIandII.

bYieldsare

basedontheaminoacidcontentoftheresinforprotected

peptides

I-X.Forprotected

peptideXIyieldswascalculatedbasedontheoreticalyield

expectedfrom

thesolution

coupling.

cSolventsystem

sare

described

intheExperim

ental.


in very large measure due to the use of the Merrifieldsolid phase method. In this special issue, devoted tothe memory of Bruce Merrifield, it is with deepgratitude that we acknowledge this debt.

EXPERIMENTAL

All reagents used were analytical grade; Boc-D-Tyr(Pri) was synthesized in this laboratory aspreviously described (Kolodziejczyk et al., 1981). Boc-Lys[Z(2Cl)]-resin was purchased from Chem-ImpexInternational (Wood Dale, IL). The b-S-(4-meth-oxybenzyl)mercapto-b,b-pentamethylenepropionic acid,Boc-Dab(Fmoc) and Z-Tyr(Bzl) were purchased fromBachem Bioscience, Inc., (King of Prussia, PA). Allother amino acid derivatives were purchased fromChem-Impex International, (Wood Dale, IL). 1-H-Pyrazole-1-carboxamidine hydrochloride was pur-chased from Aldrich, Milwaukee, WI. Thin-layerchromatography (TLC)was run on precoated silica gelplates (60F-254, E. Merck) with the following solventsystems: (a) 1-butanol:AcOH:H2O (4:1:5, upperphase); (b) 1-butanol:AcOH:H2O (4:1:1); (c) 1-buta-nol:AcOH :H2O: pyridine (15:3:3:10); (d) 1-buta-nol:AcOH:H2O (4:1:2); (e) chloroform: methanol(7:3); (f) 1-butanol:AcOH:H2O (2:1:1). Loads of10–15 lg were applied and chromatograms weredeveloped at a minimal length of 10 cm. The chlorinegas procedure for the KI-starch reagent was usedfor detection (Stewart and Young, 1984). AnalyticalHPLC was performed on a Waters 810 instrumentunder the following conditions: 90:10 to 30:70 0.05%aqueous TFA: 0.05% TFA in CH3CN, linear gradientover 30 min at 1.0 ml/min (k = 210 nm), on a Mi-crosorb C18 column (Rainin Instrument Co., Inc.). Allpeptides were at least 95% pure. Mass spectra (MS)were done by Tufts Medical School Core Facility,Medical Department, on Voyager (Applied Biosys-tems) MALDI-TOF (matrix-assisted laser desorptionionization, time of flight) mass spectrometer using di-hydroxybenzoic acid (DHB) as the matrix. Massspectra of the free peptides were in agreement with thecomposition of each peptide.

Solid Phase Synthesis Procedures

The protected precursors I–XI (Table III), of thefree peptides 1–11 (Tables I and II) were synthesizedby a combination of the Merrifield solid-phase(Merrifield 1964; Stewart and Young, 1984) with themodifications previously described (Kruszynski et al.,

1980; Manning, 1968; Manning et al., 1982, 1984,1997, 1999b) and solution synthesis methods. For thesynthesis of protected peptidyl resins, seven cycles ofdeprotection, neutralization and coupling were car-ried out starting from Boc-Lys[Z(2Cl)]-resin (Merri-field resin (chloromethylated resin) (1% cross-linkedS-DVB, 200–400 mesh, 0.5 mmol/g)]. A HCL (1 M)/AcOH mixture was used in all the deprotection steps.Neutralizations were carried out with 10% Et3N/CH2Cl2 except for the last two steps of the synthesisof peptide II, where 10% DIPEA/DCM was used.Coupling reactions were mediated mainly by DCC/HOBt (Konig and Geiger, 1970) in CH2Cl2/DMF,except for Boc-Asn which was incorporated as its 4-nitrophenyl ester (Bodanszky and Sheehan, 1964) inDMF. Protected peptide II was also obtained byMerrifield solid-phase methodology using Boc-chemistry (Manning, 1968, Merrifield, 1964; Stewartand Young, 1984; Manning et al., 1999b). To intro-duce the Nar3 residue, a Boc-Dap(Fmoc) was incor-porated in position 3. After assembly of the peptidechain the Fmoc group was removed and the peptide-resin was guanidinylated by treatment with a tenfoldexcess of 1-H-pyrazole-1-carboxamidine hydrochlo-ride as described in (Bernatowicz et al., 1992). Forprotected peptides I–IX, the acylpeptide resins werecleaved by aminolysis with Eda in MeOH (Glass anddu Vigneaud, 1973; Manning et al., 1992, 1999b). Theprotected peptide X was prepared by guanidinylationin solution of an aliquot of the protected peptide II

using 1-H-pyrazole-1-carboxamidine hydrochloride/DIPEA in DMF at 48–50�C for 6 h as described in(Bernatowicz et al., 1992). The protected retro-mod-ified precursor XI (Table III) of the free peptide 11

(Table I) was obtained by coupling the protected Edapeptide d(CH2)5(Mob)-Tyr(Pri)-Arg(Tos)-Val-Asn-Cys(Mob)-Arg(Tos)-Lys(Z(2Cl)-Eda with Z-Tyr(Bzl)in DMF using the DCC/HOBt procedure (Konig andGeiger, 1970) as described in (Manning et al., 1992).The protected peptides were purified either byextraction (for protected peptides I – IX) or by dis-solving (for protected peptides XI) with warm DMFfollowed by reprecipitations with H2O or EtOH/Et2Ountil adjudged pure by TLC, as previously described(Kruszynski et al., 1980; Manning, 1968; Manninget al., 1999b). The physicochemical properties of allprotected peptides are given in Table III.Deprotection of all protected peptides was carriedout with sodium in liquid ammonia (du Vigneaudet al., 1954a, b) as previously described (Kruszynskiet al., 1980; Manning, 1968, Manning et al., 1999b).The resulting disulphydryl compounds were oxida-

14 Manning et al.

tively cyclized with K3[Fe(CN)6] (Hope et al., 1962)using the modified reverse procedure (Rivier et al.,1978). The free peptides were desalted and purified bygel filtration on Sephadex G-15 and Sephadex LH-20,mainly in a two-step procedure (Manning et al., 1968)using 50% AcOH and 2M AcOH as eluents, respec-tively, as previously described (Kruszynski et al.,1980; Manning et al., 1999b). When necessary, anadditional purification on Sephadex G-15 or/andSephadex LH-20 with 0.2M AcOH as eluent wascarried out. The purity of the free peptides 1–11

(Table I) was checked by thin-layer chromatography(TLC), high performance liquid chromatography(HPLC) and mass spectrometry (MS).

[(b-S-(4-methoxybenzylmercapto)-b,b-pentamethylenepropionyl]-D-Tyr(Pri)-Nar-Val-Asn-Cys(Mob)-Lys[Z(2-Cl)]-Lys[Z(2-Cl)]-Eda (II, Table III)

Boc-Lys[Z(2-Cl)]-resin (0.55 g, 0.5 mmol) wasconverted to d(CH2)5(Mob)-D-Tyr(Pri)-Dab(Fmoc)-Val-Asn-Cys(Mob)-Lys[Z(2-Cl)]-Lys[Z(2-Cl)]-resin inseven cycles of deprotection, neutralization andcoupling (mediated by DCC/HOBt or active ester)with Boc-Lys[Z(2-Cl)], Boc-Cys(Mob), Boc-Asn-ONp, Boc-Val, Boc-Dab(Fomc), Boc-D-Tyr(Pri)and b-S-(4-methoxybenzyl)mercapto-b,b-pentameth-ylenepropionic acid, respectively by the manualmethod of solid-phase synthesis as previously de-scribed (Kruszynski et al., 1980; Manning, 1968,Manning et al., 1999b). The resulting protectedpeptidyl resin (1.22 g, yield 92.3%) was subjected todeprotection of the Fmoc- side chain protectinggroup at position 3 using 20% piperidine /DMF(4x5 min, 1x10 min), washed and dried. The Dab3

residue was transformed into a Nar3 residue by theguadinylation procedure (Bernatowicz et al., 1992 ) asfollows. The peptidyl resin (0.81 g) was placed in a25 ml round- bottomed flask, suspended in 5 ml dryDMF and treated with 10-fold excess of 1-H-pyraz-ole-1-carboxamidine hydrochloride(PCA)/DIPEA(0.73 g PCA/0.87 ml DIPEA) at 48–50�C for 6 hours.After the reaction was completed (negative Keisertest), the protected peptide II was obtained by ami-nolysis with ethylenediamine (Eda)/ MeOH andDMF extraction (Glass and du Vigneaud, 1973;Manning et al., 1992, 1999b). The partially protectedpeptidyl resin was placed in a 250 ml round-bot-tomed flask, 75 ml of anhydrous MeOH was added,the suspension was cooled at ca 0�C, and 30 ml Eda(99. + 5%, redistilled; Aldrich) was added with stir-ring. After 30 min, the cooling bath was removed and

the suspension stirred at room temperature for2 days. The solvents were removed on a rotaryevaporator and the protected precursor II (Table III)was extracted with warm (ca. 50�C) DMF (ca. 30 ml),and precipitated with warm (ca. 50�C) water (500ml).Following overnight storage at 4�C, the productwas collected, dried in vacuo over P2O5 to give0.61g(73.1%) of the protected acyloctapeptideEda-peptide II (Table III).

(b-mercapto)-b,b-pentamethylenepropionyl-D-Tyr(Pri)-Nar-Val-Asn-Cys-Lys-Lys-Eda (d(CH2)5[D-Tyr(Pri)2,Nar3,Val4,Lys7,Eda9]LVP (2, Tables I andII)

The Na/liq.NH3 procedure (du Vigneaud et al.,1954a,b) was used for the deprotection of protectedpeptides I–XI as described here for protected peptideII. A solution of the protected precursor II (Table -III) 150 mg, in sodium-dried ammonia (ca. 400 ml)was treated at the boiling point and with stirring withsodium from a stick of metal contained in a small-bore glass tube until a light-blue color persisted in thesolution for ca. 30 s (Manning, 1968; Kruszynskiet al., 1980; Manning et al., 1999b). NH4Cl was ad-ded to discharge the color and the ammonia evapo-rated. Reoxidation of the resulting deblockeddisulphydryl peptide obtained as a dry residue wasperformed by the modified reverse procedure Rivieret al., 1978) as follows. The disulphydryl peptideresidue was dissolved in 25 ml 50% degassed AcOHand the solution was diluted with 50 ml H2O. Thepeptide solution was added dropwise with stirringover a period of 15–30 min to an 800 ml aqueoussolution which contained 20 ml of a 0.01M solutionof potassium ferricyanide (Hope et al., 1962).Meanwhile, the pH was adjusted to approximately7.0 with concentrated ammonium hydroxide. Fol-lowing oxidation, the free peptide 2 was isolated andpurified as follows: after acidification with AcOH topH 4.5 and stirring for 20 min with an anion ex-change resin (Bio-Rad, AG 3 � 4, Cl- form, 5g dampweight), the suspension was slowly filtered and wa-shed with 0.2M AcOH (3 � 30 ml), the combinedfiltrate and washings were lyophilized. The resultingpowder was desalted on a Sephadex G-15 column(110 � 2.7 cm) eluting with aqueous acetic acid (50%)with a flow rate of 5 ml/h (Manning, 1968). Theeluate was fractionated and monitored for absor-bance at 254 nm. The fractions making up the majorpeak were checked by TLC, pooled and lyophilized.The residue was further subjected to two consecutive


gel filtrations on Sephadex LH-20 (100� 1.5 cm)eluting with aqueous acetic acid (2 and 0.2M)respectively, with a flow rate of 4 ml/min. Thepeptide was eluted in a single peak (absorbance at254 nm). Lyophilization of the pertinent fractionsgave the desired vasopressin analogue 2 (Tables I andII). With minor modifications, the same procedurewas utilized for the deprotection, cyclization andpurification of the remaining protected peptides I,

III-XI (Table III), to give the free peptides 1, 3–11

(Tables I and II).

[(b-S-(4-methoxybenzylmercapto)-b,b-pentamethylenepropionyl]-D-Tyr(Pri)-Nar-Val-Asn-Cys(Mob)-Lys[Z(2-Cl)]-Lys[Z(2-Cl)]-EdaG (X, Table III)

0.26g (0.15m mol) of protected peptide II (Ta-ble III) was dissolved in 1ml anhydrous DMF andstirred overnight with 0.22g (1.5m mol) of 1-H-pyr-azole-1-carboxamidine hydrochloride (PCA) and0.52ml (3.0m mol) of DIPEA at 48–50�C.After24 hours, TLC monitoring showed that the reactionwas still incompleted. 0.11g (0.75m mol) PCA and0.26 ml of DIPEA were added and the reactionmixture was stirred for an additional 12 hours untilthe starting protected peptide II was fully exhausted(TLC monitoring). The resulted EdaG- protectedpeptide was precipitated and washed with water,dried overnight in vacuo over P2O5, reprecipitatedfrom MeOH/ Ether, collected and dried to give 0.20g(76.9%) of protected peptide X (Table III).

(b-S-(4-methoxybenzylmercapto)-b,b-pentamethylenepropionyl]-D-Tyr(Pri)-Arg(Tos)-Val-Asn-Cys(Mob)-Arg(Tos)]-Lys[Z(2Cl)]-Eda ‹ Tyr(Bzl)(XI, Table III)

The retro-modified peptide XI (Table III) wassynthesized by a DCC/HOBt mediated coupling(Konig and Geiger, 1970; Manning et al., 1992) of theprotected Eda peptide d(CH2)5(Mob)-D-Tyr(Pri)-Arg(Tos)-Val-Asn-Cys(Mob)-Arg(Tos)]-Lys[Z(2Cl)]-Eda and Z-Tyr(Bzl) as follows. To a cooled (0�C)solution of Z-Tyr(Bzl) (0.63 g, 1.56 mmol) and HOBt(0.27 g, 2.0 mmol) in 1.5 ml of anhydrous DMF wasadded 1.2 ml (2.4 mmol) of 2M solution of DCC inDMF. The reaction mixture was stirred for 1 h,whereupon the dicyclohexylurea (DCU) was removedby filtration. The filtrate was added to a solutionof d(CH2)5(Mob)-D-Tyr(Pri)-Arg(Tos)-Val-Asn-Cy-s(Mob)-Arg(Tos)]-Lys[Z(2Cl)]-Eda (0.47 g, 0.25 mmol)in 1.5 ml anhydrous DMF. DIPEA was added to give

a pH�7.5. After the mixture was stirred for 18 h atroom temperature (TLC monitoring), methanol(20 ml) was added followed by ether (250 ml). Theprecipitated product was collected following over-night storage at 4�C. Washing with warm methanolfollowed by drying in vacuo over P2O5 gave the re-quired protected peptide XI, 1.79 g, yield 94.5%(Table III).

ACKNOWLEDGMENTS

We thank Ms. Ann Chlebowski for her expert help in the

preparation of this manuscript. This work was supported by the

National Institute of General Medical Sciences grant GM-25280.

We will be forever indebted to Bruce Merrifield for his special gift

of the solid phase method and for his friendship, advice and sup-

port over the past 40 years.

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