Peptides, Vol. 1, pp. 231-235. Printed in the U.S.A.

Bradykinin Analogs Containing :-Aminoisobutyric Acid (Aib)

R A Y M O N D J. V A V R E K A N D J O H N M. S T E W A R T

Department o f Biochemistry, Biophysics and Genetics, University o f Colorado Medical School 4200 East 9th Avenue, Denver, CO 80262

R e c e i v e d 18 June 1980

VAVREK, R. J. AND J. M. STEWART. Bradykinin analogs containing oeaminoisobutyric acid (Aib). PEPTIDES 1(3) 231-235, 1980.--All seven possible bradykinin (BK) analogs containing Aib in place of proline have been synthesized by the solid phase method and assayed for in vitro myotropic activity on the guinea pig ileum and rat uterus, and in rivo on the rat blood pressure, both by intravenous and intra-aortic administration. [AibZ.3]-BK, [Aib2.7]-BK, and [Aib2':~'r]-BK had no in vivo or in vitro activities; [AibZ]-BK, [Aib'~]-BK and [Aib3'r]-BK had moderate BK-like activities and a significantly increased resistance to pulmonary inactivation in the rat ([Aib3'r]-BK was totally resistant). [Aibr]-BK was found to be the most active position seven BK analog yet assayed on the rat blood pressure, and shows remarkably high ileum 4 times BK) and intravenous rat blood pressure (6 times BK) activity.

Bradykinin analogs a-Aminoisobutyric acid (Aib)

THE search for an understanding of how small physiologi- cally active peptides interact with their biological receptors has taken many forms. Since the demonstration that low molecular weight peptides could be routinely synthesized [34], and since the advent of a rapid and reliable method of peptide synthesis [18], the judicious design [26] and synthe- sis of several thousand analogs of many biologically impor- tant peptides have led to some insight into how the individual elements of a peptide (i.e., primary sequence, functional groups, backbone and side chain conformation, overall molecular topography) contribute to receptor binding and biological response.

We were interested in the introduction of elements into the bradykinin sequence (Arg-Pro-Pro-Gly-Phe-Ser-Pro- Phe-Arg) which would alter the conformational possibilities available to the peptide molecule. This in itself would not be a sufficient goal, since any restricted conformation analog which lacked bradykinin-related biological activity would severely limit the useful conclusions which could be made about receptor binding conformations. It was therefore of prime importance that the method used to restrict the con- formation also allow for one or more of the permitted con- formations to react with bradykinin receptors in biological systems, and provide analogs with either agonist or antagonist activity.

Bradykinin (BK) exists in solution as a population of con- formations which can be considered to be partially ordered [4] or primarily disordered [16], depending on the nature of the solvent and the method used for obtaining the structural information. The three proline residues in BK, and espe- cially the Pro2-Pro 3 sequence, should restrict conformational possibilities, but strong spectral evidence of this has not been seen [16]. A recent report that a model tetrapeptide containing the Pro-Pro sequence can adopt a 310 helical con- formation [33] and several reports that the c~-aminoisobutyric

acid (Aib) residue in peptides shows a similar strong tend- ency to induce both/3-turns and incipient 31o helical structure [3, 20, 23, 24, 29] suggested that the Aib residue would be a prime candidate for substitution into BK in place of the proline residues. Aib substitution has been used in studies of the biological conformations of angiotensin-II [ 12,17], and is found in nature as a constituent of the peptide ionophore alamethicin [19] and related antibotics [8, 13, 21, 22].

METHOD

Peptide Synthesis

All of the peptides were synthesized by the solid phase method [18] by use of a Beckman 990 Peptide Synthesizer (Beckman Instruments, Palo Alto, CA) using standard pro- cedures [30]. Boc-Aib (see reference 110] for the standard IUPAC abbreviations) was made from Aib (Sigma Chemical Company, St. Louis, MO) with 2-tert-butoxycarbonyioxyimino- 2-phenylacetonitrile (BOC-ON ®, Aldrich Chemical Com- pany, Milwaukee, WI) [9]. The remaining protected amino acids (Boc-Arg(Tos), Boc-Pro, Boc-Gly, Boc-Phe, Boc-Ser (OBzl)) were commercially available (Bachem, Torrance, CA, and Beckman, Palo Alto, CA). Coupling reactions were monitored by use of the Kaiser test [14]. Standard coupling reactions used 2.5-fold amounts of Boc-amino acids and di- cyclohexylcarbodiimide. Boc-Aib and the next Boc-amino acid following Aib were recoupled with the same amounts of reagents, and the peptide-resins were then acetylated at these points with acetic anhydride and triethylamine. Pep- tides were cleaved from the resin by anhydrous HF in the presence of anisole [27] and purified by countercurrent dis- tribution (100 upper phase transfers in the system n-BuOH:l% T F A (1:1)). Peptide purity was determined by TLC (Merck silica gel coated glass plates) in two different

C o p y r i g h t © 1980 A N K H O I n t e r n a t i o n a l Inc . - -0196-9781/80/030231-05501.00/0

232 VAVREK AND STEWART

solvent systems (nBuOH:AcOH:H20 - 4:1:1, and EtOAc; Pyr idine:AcOH:H20 - 5:5:1:3), and high voltage paper elec- trophoresis (10 V/cm on Whatman No. 1 paper) in N AcOH (pH 2.8); peptides were visualized with the Sakaguchi spray for arginine and the chlorine/tolidine spray for the peptide bond. Peptide identity was confirmed by quantitative amino acid analysis (Beckman 120 C) after acid hydrolysis in sealed glass tubes under N_,.

Bioassay

The in vitro guinea pig ileum [321 and rat uterus ]6] assays were used to determine BK-like myotropic activity and to check the BK antagonism. The in vivo rat blood pressure assay [25] was performed in such a way that the amount of inactivation of the peptide upon passage through the pulmo- nary circulation could be determined by the difference in the depressor effect after intravenous (IV) and intra-aortic (IA) administration. In our assays, the amount of BK inactivation varied between 96--99%.

RESULTS

Some physical properties of the Aib-BK peptides are given in Table 1. Acid hydrolyzates of all the peptides showed the expected amino acid ratios within experimental error.

Table 2 contains the bioassay data for the seven Aib analogs, along with data reported for some other BK analogs having substitutions in positions 2, 3, and 7 [28,31]. No anti-BK activity was seen with the Aib analogs in any assay.

DISCUSSION

Some difficulty was expected in the coupling of Boc-Aib by the solid phase method in light of the reports of difficulty in the solution synthesis of Aib-containing peptides [11,15] and because the solid phase synthesis of alamethicin re- quired very long reaction times for complete addition of Boc-Aib [7]. It was expected, however, that the coupling of Boc-Aib to the growing peptide chain and the coupling of subsequent Boc-amino acids to the N-terminal Aib residue could be monitored with the Kaiser qualitative ninhydrin reagent [14], since Aib had been reported to react with ninhydrin during quantitative amino acid analysis [7], albeit with a very low color yield (about 10% of the average color values for the standard amino acids). However, the reactiv- ity of the deprotec ted and neutral ized Aib-terminal pep- tide resins toward the Kaiser reagents was unpredictable, and a very posi t ive (deep purple color) or total ly negative (pale yel low-green color) test was seen with closely related Aib-terminal pept ide resins. Thus, Aib-Arg(Tos)-O-Resin, Aib-Pro-Gly-Phe-Ser(Bzl)-Pro-Phe-Arg(Tos)-O-Resin, and Aib-Gly-Phe-Ser(Bzl)-Aib-Phe-Arg(Tos)-O-Resin gave the normal positive ninhydrin color, while Aib-Gly-Phe-Ser (Bzl)-Pro-Phe-Arg(Tos)-O-Resin, Aib-Phe-Arg(Tos)-O-Res- in, Aib-Aib-Gly-O-Resin and Aib-Aib-Gly-Phe-Ser(Bzl)- Aib-Phe-Arg(Tos)-O-Resin gave a totally negative Kaiser test. In view of this inability to monitor Aib coupling re- act ions, Boc-Aib and each Boc-amino acid immediately fol- lowing Aib were double coupled and the pept ide-resin then acetylated to block any unreacted terminal amino groups. With these precaut ions the overall yields and purity of the Aib-BK analogs were no different than those regularly obtained for BK analogs in this laboratory. Compared with

TABLE 1 PHYSICAL PROPERTIES OF AIB PEPTIDES

TLC-~ Peptide k (CCD)* HVE¢ 411 5513

BK 1.13 0.71 0.02 - - [AibZ]-BK 1.63 0.73 0.02 0.29 [Aib:~I-BK 1.70 0.79 0.02 0.30 [AibT]-BK 1.50 0.72 0.02 0.29 [Aibe':q-BK 3.76 0.74 0.03 0.46 IAib2'TJ-BK 2.33 0.72 0.02 0.30 [Aib:~'r]-BK 2.57 0.72 0.04 0.41 [Aib'-":~'7]-BK 4.88 0.72 0.04 0.41

*The experimentally determined partition coefficient from counter- current distribution in the system n-BuOH:l% TFA (1:1).

tMobilities relative to Lys during high voltage paper elec- trophoresis in N AcOH (pH 2.8).

SR~ values from thin layer chromatography on Merck silica gel coated glass plates in the systems 411 (n-BuOH:AcOH:H20--4:1 : 1) and 5513 (EtOAc:Pyridine:AcOH:HzO~5:5:1:3).

BK, all of the Aib analogs showed an increased hydrophobic character as reflected by the partition coefficients deter- mined for them during their purification by countercurrent distribution in n-BuOH: 1% TFA (1: 1). In order of increasing hydrophobicity they are BK(k= I. 13), [Aibq-BK, [Aib2]-BK, [Aib3]-BK, [Aib2'7]-BK, [Aiba'7]-BK, [Aib2'a]-BK and [Aib2"3,7]-BK. We have found (unpublished results) that in all cases the mono-substituted D-Pro-BK analogs have even higher partition coefficients than the mono-Aib analogs. However, the tri-substituted Aib analog has a significantly higher k than the tri-substituted D-Pro analog (4.8 vs 3.2), indicating that the three Aib residues, and especially the Aib2-Aib 3 sequence, might be causing the molecule to as- sume a more tightly packed form which is less hydrophilic than the D-Pro analog. The increased hydrophobicity of D-Pro bradykinins over bradykinin itself must be due to al- tered peptide conformation, and the effect of the Aib sub- stitutions is most likely also due to an altered peptide con- formation. If the biological activities found for the Aib analogs also mirror peptide conformation, it is not surprising that the highest BK-like activity was found for [Aibq-BK, whose partition coefficient is closest to that of BK (most hydrophilic).

Due to the lack of complete bioassay data in the literature for most of the BK analogs which have been reported, it is possible to discuss the activities of the Aib analogs only in comparison with a few D-Pro and L-Ala substituted BK analogs. [Aib2]-BK shows a resistance to pulmonary inac- tivation similar to that of [D-Proe]-BK (65%, vs 8 ( ~ reported for the D-Pro analog), and about 1/3 of the IA blood pressure activity, which mirrors the relative myotropic potencies for these two peptides. [Aib'~]-BK is some 600 times more active than the D-Pr& analog, but 100 times less active than [Ala3]-BK, which is equipotent with BK in all of the assays, including susceptibility to pulmonary inactivation. [Aiba]- BK is more resistant to this degradation than BK and [AIa3]-BK, but does not approach the 30% inactivation of [D-Pro3]-BK. This indicates that Aib in position 3 of BK may have enough D-amino acid quality to prevent some of the metabolic processing which could deactivate the peptide by

a - A M I N O I S O B U T Y R I C B R A D Y K I N I N A N A L O G S 233

T A B L E 2

BIOASSAY DATA FOR AIB-BK PEPTIDES AND RELATED ANALOGS*

In vitro¢ In vivo$ Peptide GPI RUT RBP (IV) RBP (IA)

Pulmonary destruction§ (percent)

BK 100 100 100 100 96-99 [Aib2]-BK 0.3 0.5 2.5 0.07 65 [Aib:~]-BK 3.3 7.7 24 1.2 80 [Aibr]-BK 400 67 570 43 50 [Aib2':~]-BK 0 0 0 0 - - [Aib2'r]-BK 0 0.03 0 0 - - [Aib:~'r]-BK 0.25 1 0.7 11 0 [Aib2':~'7I-BK 0 0 0 0 - - [Ala2]-BK 0.7 0.3 - - - - - - [D-Pro~]-BK - - 0.2 - - 0.2 - - [VaF]-BK 0.2 1 - - - - - - [Sare]-BK 40 . . . . [APro2]-BK¶ 93 120 90 - - - - [Ala'~]-BK 100 100 - - 120 97 [GIy:q-BK 14 . . . . [D-Pro:q-BK - - 0.01 - - 0.002 30 [VaV]-BK 0.07 . . . . ISafq-BK - - 67 - - - - - - [APro:q-BK 82 97 71 - - - - [Alar]-BK 1 1 - - - - - - [GlyT]-BK - - 1 - - - - - - [D-Pro7]-BK - - 1 - - 0.5 0 [Sar7]-BK 13 . . . . [APror]-BK 12 25 23 - - - - [D-Pro2"~]-BK - - 0.01 - - - - - - [Sar2':~]-BK 5 . . . . [D-Pro2'7]-BK - - 0.01 - - - - - - [D-Pr&'7]-BK - - 0.001 - - - - - - [D-Pro2':I'7]-BK - - 0.0005 - - - - - - [ SarZ':~'7]-BK 1 . . . .

*See references [8] and [31] for non-Aib BK analogs listed in the table. ?Myotropic activity determined on the guinea pig ileum (GPI) and rat uterus (RUT) [6] [32]. ~;ln vivo rat blood pressure depressor effect after intravenous (IV) and intraaortic (IA) admin-

istration [25]. §% Destruction= 100-[(EDsoAI)/(ED~,IV)] × 100 [25]. ¶L-(3,4-dehydro)-Pro.

c leaving the Pro3-Gly 4 bond . It is known that a-alkyl amino acids in pept ides confer res i s tance to various pep t idases [1, 2, 5], and this res i s tance to enzymat i c a t tack could be seen as coming f rom the partial D-nature of the Aib res idue. In addi- t ion to its ability to restr ict the backbone confo rma t ion o f pept ides [3], the Aib res idue could appear as bo th a D-Ala and an L-AIa at the same t ime to a biological recep tor .

A n o t h e r example of the D- and L- quali ty of the Aib resi- due might be seen in [Aibr]-BK. The D-Pro 7 analog is totally res is tant to pu lmonary b r e a k d o w n , while the Aib 7 analog shows 50% des t ruc t ion . It is known that the ProT-Phe 8 bond of BK is c leaved in the lung, mos t p robably by the pept idyl d ipept idase (angiotensin conver t ing enzyme , E.C.3.4.15.1) [35,36], and the total inhibit ion o f this part icular c leavage by the incorpora t ion of a D-amino acid (D-Pro), or the partial inhibit ion by a " p s e u d o - D " amino acid (Aib), could account for part o f the obse rved res i s tance to inact ivat ion. The re- s is tance of [AiW]-BK to enzymat i c degradat ion undoubted ly accounts for some of its very significant agonist act ivi ty in

the assays , especial ly the ileum, where it is 4 t imes as potent as BK, and in the IV blood pressure assay where it is near ly 6 t imes as potent . But even in the assays in which kininase b r e a k d o w n is not general ly a major factor , [AibT]-BK shows remarkably high activity. With 43% of the po tency of BK on the IA rat b lood pressure , [AibT]-BK is the mos t act ive posi- tion seven analog ever repor ted . It is unfor tunate that com- parable b lood p ressure data are not available for many of the posi t ion seven analogs listed in Table 1 so that the very intriguing proper t ies of the Aib res idue could be examined more thoroughly.

Ne i the r [Aib2.3]-BK nor [Aib2 'q-BK showed any rat blood p ressure dep re s so r effect , nor any an tagonism o f the re- sponse to BK. E x cep t for a very slight uterine effect of the Aib 2,T analog (0.03%) they are also inactive in the myot rop ic assays , [Aib3a]-BK (which co mb i n es the subst i tu t ion o f Aib in two posi t ions which give, individually, modera t e (Aib 3-) or exce l len t (Aib 7-) activity in all o f the assays , and good resist- ance to pu lmonary inactivation) shows comple te res i s tance

234 V A V R E K A N D S T E W A R T

to p u l m o n a r y b r e a k d o w n and a b o u t 1% of B K act iv i ty in the u te rus and in the in t ra -aor t ic b lood p re s su re assays . The t r i subs t i t u t ed ana log [Aib2,3,7]-BK was devo id o f any BK-l ike ac t iv i ty , wh ich is p r o b a b l y not surpr is ing. H o w e v e r , [AibZ,:~,7]-BK shares wi th [Aib2,3]-BK an abil i ty to e n h a n c e sl ightly the r e sponse of s m o o t h musc le to BK. The na tu re of this po ten t i a t ion , wh ich has not b e e n repor ted for any o the r B K analog, is u n k n o w n .

T a k e n by t h e m s e l v e s , the biological da ta on the A i b - B K ana logs do not yield a comple t e u n d e r s t a n d i n g o f the na ture or magn i tude of the con t r i bu t i on of a l te red pept ide con-

fo rma t ion to the BK-l ike biological r e sponses . A more thor- ough u n d e r s t a n d i n g of the con fo rma t iona l aspec t s mus t awai t comple t ion of the so lu t ion c o n f o r m a t i o n s tudies now u n d e r way on these pept ides .

ACKNOWLEDGEMENTS

The authors thank Ms. Li-Hsueh Hsi for performing the bioas- says, and Ms. Virginia Sweeney for the amino acid analyses. This work was supported by NHLBI-NIH Contract No. 1-HV-72946.

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