Vol. 54 No. 4/2007, 805–811 Regular paper on-line at: www.actabp.pl

Eleven oxytocin analogues substituted in position 4, 5 or 9 by tetrazole analogues of amino acids were prepared using solid-phase peptide synthesis method and tested for rat uterotonic in vitro and pressor activities, as well as for their affinity to human oxytocin receptor. The tetrazolic group has been used as a bioisosteric substitution of carboxylic, ester or amide groups in structure-activity relationship studies of biologically active compounds. Replacement of the amide groups of Gln(4) and Asn(5) in oxytocin by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities. Oxytocin analogues in which the glycine amide residue in position 9 was substituted by tetrazole analogues of glycine had diminished activities as well. The analysis of differences in rat uterotonic activity and in the affinity to human oxytocin receptors of analogues containing either an acidic 5-substituted tetrazolic group or a neutral 1,5- or 2,5-tetrazole nucleus makes it possible to draw some new conclusions concerning the role of the amide group of amino acids in positions 4, 5 and 9 of oxytocin for its activity. The data suggest that the interaction of the side chain of Gln(4) with the oxytocin receptor is influenced mainly by electronic effects and the hydrogen bonding capacity of the amide group. Steric effects of the side chain are minor. Substitution of Asn(5) by its tetrazole derivative gave an analogue of very low activity. The result suggests that in the interaction between the amide group of Asn(5) and the binding sites of oxytocic receptor hydrogen bonds are of less importance than the spatial requirements for this group.

We present here the synthesis and biological activities of 11 oxytocin analogues, in which the carboxamide group in positions 4, 5 or 9 was substituted by the bioisosteric tetrazolyl group (Butler, 1984;Burger, 1991;Herr, 2002).5-Substituted tetrazoles, frequently referred to as tetrazolic acids, behave as acids comparable in character to carboxylic acids.Their pK a values are in the same range as those of carboxylic acids (Herbst & Wilson, 1957;Kaczmarek, et al., 1979).Therefore, they are ionized at a physiological pH.5-Substituted tetrazoles exist as 1H-and 2H-tautomers (Fig. 2).Tetrazolic acids and especially tetrazolate anions have high capacity for hydrogen bond formation in the interaction with other molecules (Rażyńska et al., 1983).Hansch and Leo reported that tetrazolate ions are almost 10 times more lipophilic than the corresponding carboxylic anions (1995).This property and the high metabolic stability of tetrazolyl group are important factors for the design of potential drugs.Considering the structure of 5-substituted tetrazoles and their N 1 -or N 2 -substituted derivatives, they can mimic esters and amides as well (Fig. 2).Therefore, tetrazoles can be used in order to elucidate the role of hydrogen bonds formed by amide groups in the interaction of biologically active peptides with their receptors.
The aim of our study was to use the bioisosteric tetrazolyl group in the hope of increasing our understanding of the role of amide groups of OT for the biological functions of the hormone.Therefore, we have substituted the Gln 4 , Asn 5 and Gly-NH 2 9 residues in OT by their tetrazole derivatives (Fig. 3) and studied biological properties of the resulting analogues.

EXPERIMEnTAL PROcEduRES
Materials.Fmoc-amino acids and CLEAR-resin were purchased from Peptides International, Inc. Syntheses of Fmoc-derivatives of tetrazole analogues of glycine, α-aminoadipic (racemic or l-enantiomer), aspartic and glutamic acids have been reported recently (Manturewicz et al., 2007;Manturewicz & Grzonka, 2007).TLC analysis was carried out on aluminium sheets precoated with silica gel 60, F-254 (Merck).RP-HPLC used for purification of products and for their analyses was performed on Kromasil C4 or C8 columns. 1 H-NMR spectra were measured on a Varian Mercury 400 MHz spectrometer.IR spectra were measured on a Bruker IFS66 spectrophotometer.Mo-   lecular weights of the compounds were determined on a Bruker BIFLEX III MALDI-TOF spectrometer.
Peptide synthesis.Oxytocin analogues were synthesized manually by solid-phase method on CLEAR-amide resin.[1-7]OT was synthesized on 2chlorotrityl-resin.The following side chain protecting groups were used: tert-butyl for Tyr and trityl for Asn, Gln and Cys.Deprotection of Fmoc group was carried out using 20% piperidine/DMF (1 × 5 min, 1 × 20 min).The resin was washed subsequently 3 times with DMF, 3 times with dichloromethane, and once with DMF.Deprotection and couplings were monitored using the Kaiser ninhydrin test (Kaiser et al., 1970).Coupling was performed in DMF with 3 equiv.of Fmoc-amino acids and 3 equiv.of TBTU or DIPEA.Coupling time was 2 h.In the case of Fmoc-Asp(T 2 CH 3 )-OH, Fmoc-Glu(T 1 CH 3 )-OH and Fmoc-L-Aaa(T)-OH, due to small amounts of these derivatives available, couplings were performed with 1 equiv.for 6 h.Free amino groups which were not acylated during couplings were blocked with the use of N-acetylimidazole.After completion of the synthesis, the peptidyl-resin was washed stepwise with DMF, methanol and ethyl ether and dried.The peptide was cleaved from the resin using a cocktail of TFA, TIPS, phenol and water (8.8 mL: 0.2 mL: 0.5 mL: 0.5 mL per g of the resin, respectively) for 2 h at room temp.under argon atmosphere with agitation.The cleaved resin was then filtered off, and the resultant solution was concentrated in vacuo.The crude oxytocin analogue was precipitated with cold ethyl ether.The ether layer was decanted after 15 min of centrifugation.This process of adding ether, centrifugation and decanting was repeated 5 times and the product was dried by stream of argon.The peptide was oxidatively cyclized with 0.1 M I 2 in MeOH using the standard procedure (Atherton & Sheppard, 1989).Then Dowex 1 × 8 (CH 3 COO -form) was added to the solution under stirring.After 20 min, the supernatant was removed, the solvent evaporated under reduced pressure, and the residue dissolved in water and lyophilized.The crude product was purified by RP-HPLC.When racemic Fmoc-Aad(T)-OH was used in the synthesis of appropriate oxytocin analogue, two diastereoisomeric peptides were obtained, which were separated by RP-HPLC.The separated [Aad(T) 4 ]OT was found to be identical with the oxytocin analogue synthesized from the l-enantiomer of Fmoc-Aad(T)-OH.The purity and identity of each oxytocin analogue was determined by analytical RP-HPLC and MALDI-TOF mass spectrometry.
Biological evaluation.Wistar rats were used in all experiments.Synthetic oxytocin and argininevasopressin were used as standards in the uterotonic test and pressor test, respectively.The uterotonic activity was determined in vitro on an isolated strip of rat uterus in the absence or presence of magnesium ions (Holton, 1948;Rudinger & Krejči, 1962).In principle, cumulative dosing was applied in most experiments, i.e. doses of standard or of the analogue were added successively to the uterus in the organ bath in doubling concentrations and at 1 min intervals without the fluid being changed until the maximal response was obtained.Each analogue was tested using uteri from 3-5 different rats.Pressor activity was determined on phenoxybenzaminetreated male rats in urethane anesthesia (Dekansky, 1952).The responses to standard doses of oxytocin or vasopressin were stable for several hours, with-out problems with tachyphylaxis.For more details concerning the tests see Slaninova (1987).
Binding affinity determination.Determination of binding affinity to human oxytocin receptor (OTR) was performed as described by Fahrenholz et al. (1984) using tritiated oxytocin from NEN Life Science (Boston, MA, USA).In brief, a crude membrane fraction of HEK OTR cells, i.e.HEK cells with stable expression of human OT receptors, kindly donated by Dr. G. Gimpl (Gimpl et al. 1997), was incubated with [ 3 H]OT (2 nM) and various concentrations of peptides (0.1-10 000 nM) for 30 min at 35 o C. The total volume of the reaction mixture was 0.25 mL.Buffer used was 50 mM Hepes at pH 7.6 containing 10 mM MnCl 2 and 1 mg/mL bovine serum albumin.The reaction was terminated by quick filtration on a Brandel cell harvester.Binding affinities were expressed as IC 50 values calculated from the binding curves using GraphPad Prism 3.02.

RESuLTS And dIScuSSIOn
Recently we have worked out methods of synthesis of Fmoc derivatives of tetrazole derivatives of Asp, Glu and Aad, compounds suitable for solid-phase peptide synthesis (Manturewicz & Grzonka, 2007b).Now, using these compounds we present manual solid-phase peptide synthesis of eleven new analogues of OT (I-XI) (Table 1) on a CLEAR-amide or 2-chlorotrityl resin following Fmoc chemistry (Atherton & Steward, 1989).All the analogues contain tetrazole derivatives of amino acids in which the tetrazole ring is either in the side chain of appropriate amino-acid residues (derivatives of aspartic, glutamic or α-aminoadipic acids) or in the C-terminal position.The amide groups of glutamine in position 4 (analogues I, V, VIII and IX), asparagine in position 5 (analogue IV) or glycine amide in position 9 (analogue X) were substituted with acidic 5-tetrazolyl group.Other analogues (II, III, VI, VII and XI) contain N-methylated tetrazole ring (at N 1 or N 2 atoms) and due to this modification the ring has not acidic properties, but on the other hand, it still retains the ability to form hydrogen bonds.The oxytocin analogues containing tetrazole derivatives of aspartic, glutamic and α-aminoadipic acids were obtained by stepwise coupling of Fmoc-amino acid to the growing peptide chain, whereas the analogues in which the Gly-NH 2 residue in position 9 of OT was substituted by tetrazole derivative of glycine (X and XI) were synthesized by fragment condensation.
First, H-Leu-GlyT and H-Leu-GlyT 1 CH 3 were synthesized by different methods (see Experimental), and then they were coupled to the [1-7]OT fragment.The purity of all oxytocin analogues prepared was higher than 99% and their structures were confirmed by MS (Table 1).The analogues were tested for their rat uterotonic in vitro and pressor activities and for their affinity to human oxytocin receptor.The data of the bioassays are presented in Table 2.
Replacement of the amide groups of Gln 4 , Asn 5 and Gly-NH 2 9 by tetrazole groups led to analogues I-XI with low uterotonic activity in comparison to the parent hormone (Table 2).The potency of these analogues increased 2 to 15 times when it was determined in the presence of Mg 2+ ions.The enhancing effect of Mg 2+ ions (Munsick, 1960;Krejči & Polaček, 1968) occurs at the receptor level and is not a property of the peptide per se (Soloff & Grzon-ka, 1986).Analogues in which Gln 4 was substituted with tetrazole derivatives of aspartic, glutamic or α-aminoadipic acid (analogues I, V and VIII) had approx.2-4 % or 4-30% of the uterotonic activity of OT in the absence of Mg 2+ or in the presence of Mg 2+ ions, respectively.Analysis of these data revealed that the replacement of amide group by the acidic tetrazolic group is the main reason of the diminished activities of the analogues.The steric effect of the tetrazole group is less important, because the analogues I, V and VIII, which differ in the length of the side chains in position 4, have roughly similar uterotonic activities.The data presented here thus suggest that the interaction of the side chain of Gln 4 with the OTR is influenced mainly by electronic effects and hydrogen bonding capacity of the carboxamide group rather than by steric effects of the side chain.
N-methylation of the tetrazole ring of the amino-acid residue in position 4 lessened further the uterotonic activities in comparison to the nonalkylated analogues.In the case of N-methylated Asp(T) 4 analogues, higher activity was found for the analogue methylated on the N 2 tetrazole atom (compound III), whereas with analogues containing an N-methylated Glu(T) residue more active was the analogue methylated on the N 1 atom (compound VI).Generally, OT analogues having N-methylated tetrazole derivatives of Asp were more active than the corresponding Glu analogues.
As one could expect, substitution of Aad(T) in position 4 by its d-enantiomer (compound IX) led to the analogue which was almost inactive.
The lowest uterotonic activity was found for analogue IV that contains the tetrazole derivative of aspartic acid in place of asparagine in position 5.This finding shows that the introduction of the acidic tetrazolic group in this position substantially changes the interaction of the side chain of the appropriate amino-acid residue with oxytocic receptor and confirms the crucial role played by the amide group of Asn 5 in OT structure.Replacement of glycine amide in position 9 by a Gly(T) residue also gave analogue with very low uterotonic activity.In this case N-methylation did not result in a decrease of the activity.On the other hand, the N 1 -methylated GlyT analogue (compound XI) showed uterotonic activity three times higher than the [GlyT 9 ]OT (X) analogue.The activity of the two latter analogues was not enhanced in the presence of magnesium ions.
There is a good correlation between the uterotonic activity of an analogue measured in the presence of Mg 2+ ions and its affinity to human oxytocin receptor (Table 2).For analogues I, II, III, V and VIII the affinities for OTR are approx.10 times lower than that of the parent hormone.Again, substitution of asparagine in position 5 with the tetrazole analogue of aspartic acid gave analogue IV with a very low affinity to OTR.
Only analogues of oxytocin substituted in position 4 by the α-aminoadipic acid residue had residual pressor activity.All other analogues were devoid of pressor activity.
The tetrazole derivatives of Asp, Glu and Gly proved useful in explaining the role of amide groups in the structure-activity relationships of oxytocin.Therefore, they can be useful also in the SAR analysis of other peptides containing asparagine, glutamine or glycine amide.

Figure 3 .
Figure 3. Tetrazoles analogues of amino acids in this study.