Int. J. Peptide Protein Res. 39, 1992, 401-414

Reduced peptide bond cyclic somatostatin based opioid octapeptides Synthesis, conformational properties and pharmacological characterization WIESLAW M. KAZMIERSKI', RON D. FERGUSON ', RICHARD J. KNAPP', GEORGE K. LU12, HENRY I. YAMAMURA' and VICTOR J. HRUBY

'

Department of Chernistiy, University of Arizona, Department of Phnrmcicology, Arizona Health Sciences Center, Tucson, Arizona, USA

Received 6 June, accepted for publication 20 November 1991

The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the p opioid receptor selective antagI

I

(IV), have been investigated. Peptide analogues onist ~-Phe~-Cys~-Tyr~-~-Trp~-Orn~-Thr"-Pen~-Thr~-NH2 that contain [CH,NH] and [CHzN] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinolinecarboxylate (Tic) and of D-Trp" with D- 1,2,3,4-tetrahydro-~-carboIine(~-Tca~), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for p opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations I

1

of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-ThrNH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the p opioid receptor. Peptide bond modifications were CH~NI-D-TC~'), 2; and (Cys2-$[ CH2N]-Tic3)),6. made including (Tyr3-$[ C H ~ N H I - D - T ~ ~3; " )(Tyr3-$[ , These analogues showed decreases in their p opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 'H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between p and 6 opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility. Key words: CH2NR>(R# H ) amide bond surrogate; conformation; p opioid receptors; NMR; opioid peptides; reduced peptide bonds; tetrahydrocarboline

In earlier work (1-3) we have developed an approach (referred t o a s "Topographical design on a stable template") t o control peptide topography by utilizing

unusual, side chain-conformationally biased amino acids in the peptide sequence, but which still retain the backbone conformation of the peptide. For this purpose we have designed several somatostatin-derived octapeptide opioid antagonists with specific topographical features (e.g. I, 11, 111, and IV, Table l), and examined their ability to recognize (bind to) either p or 6 opioid receptors (2,3). F o r example, replacement of

Abbreviations of the common amino acids and their derivatives are in accordance with the recomniendations of IUPAC-IUB (J. Biol. Chmi. 264, 668-673, 1986). Additional abbreviations include: Tic, 1,2,3,4-tetrahydroisoquinolinecarboxylate; Tca, 1,2,3,4-tetrahydroD-Phe' in I by D- 1,2,3,4-tetrahydroisoquinoline carboxP-carboline; TLC, thin-layer chromatography; H PLC, high perforylate (D-Tic) resulted in a substantial increase in the mance liquid chromatography; FAB-MS, fast atom bombardment selectivity and affinity of I1 for p opioid receptors (2). mass spectrometry; pMBHA, p-methylbenzhydrylamine; TFA. tri'H NMR conformational analysis showed that the fluoroacetic acid; BOP, bcnzotnazol-I-yloxy-tris-(dimethyl-amino)phosphonium hexafluorophosphate; and DCM, dichloromethane; N-terminal D-Tic' residue has exclusively a g( + ) side LAH, lithium aluminum hydride. chain conformation (3-5), resulting in an arrangement

401

W.M. Kazmierski et ul. of aromatic side chains of D-Tic' and Tyr3 on the same face of the molecule (1, 3). This topography could be altered, resulting in an opposite arrangement (mismatch) of these aromatic moieties (3). by acylation of the N-terminal D-Tic with Gly (111, Table l), leading to the gciuche( - ) side chain conformation of D-Tic'. 111 showed a large decrease in affinity for p opioid receptors, and a modest increase of affinity for 6 receptors (2). Thus, we concluded that the N-terminal D-Tic prefers a gauche( + ) side chain conformation, whereas when a D-Tic residue is in the peptide chain, it prefers a gauche( - ) side chain Conformation. In our efforts to further understand the conformational properties of pipecolic acid systems such Tic, and their possible application in topographical design, we have turned our attention towards replacement of the peptide bond, centered on the pipecolic acid amino group, by an aminomethylene group. We reasoned that the strong 1,2 diequatorial interactions of the N- and C-substituents present in the gauche( + ) conformation (Fig. 1. II), that cause the gauche( - ) conformation to be more stable for an acylated amino group Tic (3, 6), could be relieved if the N-substituent (CH2-N-C,) were forced to attain an out of plane conformation. To achieve this, the properties that cause its planar arrangement (resonance of the carbonyl group and the free electron pair of the amino group) needed to be modified. In principle, this can be done by replacing the carbonyl group preceding the pipecolic acid amino with a methylene unit. If, indeed, the resulting sp3 hybridization allows for nonplanarity of both the C- and N-substituents, then it is possible that a 1,2-diequatorial relation may be energetically tolerated by the molecule in a gccuche( + ) side chain conformation. The required alkylamine may be obtained synthetically by reductive alkylation of a pipecolic acid derivative with an amino aldehyde in the presence of sodium cyanoborohydride. This procedure

I

I1 111

IV 1

2

3

5

402

has been successfully used to obtain CHzNH units both in a solution (7) and on solid supports (8), with a goal to increase peptide resistance to proteases (9). To our knowledge, CH2NR2 (where R # H) units have not yet been constructed in peptide syntheses, particularly on solid supports. In this paper we present a series of six peptides (1-6, Table l), including four with modified peptide bonds, that address the above design questions. We have synthesized analogues of peptide I (with Orn5 replacing Lys5)and withTic' or D-Tca4(Tca = 1,2,3,4-tetrahydroP-carboline) (peptides 4 and 1, respectively, Table 1). Thus, the peptide bonds preceding the pipecolic acidderived amino acids in the peptide chain were replaced by either CH2N (peptides 2, 5 , 6 ) or CHzNH (in a control peptide 3). We also present the pharmacological properties of these new peptides and their conformational properties. MATERIALS AND METHODS

Geiieral methods Syntheses of peptides 1 through 6 were accomplished by methods reported previously (1, 2) utilizing standard solid phase synthetic techniques (10, 11) on a Vega (Tucson, AZ) Model 250 or 1000 peptide synthesizer. N"-Boc protected amino acids were either purchased from Bachem (Torrance, CA) or were prepared by literature methods (1 1). Carboxamide terminal peptides were synthesized using a p-rnethylbenzhydrylamine (pMBHA) resin that was prepared by literature methods (12); resin substitution was 1.0 mM/g. A 1.5 M excess of preformed symmetrical anhydrides or a 3 M excess of N-hydroxybenzotriazole active esters was used for coupling reactions, which were monitored by ninhydrin (13) or chloranil tests (14). Purity of the final peptides was assessed by TLC in four different solvents, RP-HPLC, FAB-MS (Table 2), and 'H NMR. Capillary melting points were determined on a ThomasHoover apparatus and are uncorrected. Purity for each TABLE I amino acid was established by the ninhydrin test, 'H Chemicul sfrucrure.T of .sytherir opioid peprides NMR, optical rotation (sodium D line, Rudolph Research Auto-Pol 111 polarimeter), and TLC. Purification of peptides was accomplished by a combination of D-Phe-Cys-Tqr-D-Trp-Lqs-Pen-Thr-,VHH(CTP) gel filtration, partition chromatography and reversed D-Tic-Cys-Tyr-D-Trp-Lys-Pen-Thr-,~H-(TCTP) phase high performance liquid chromatography (RPI I Gly-D-Tic-C)s-Tyr-D-Trp-Om-Thr-Pen-Thr-hH_. HPLC). For most cases gel filtration (G-15) followed I I by RP-HPLC was sufficient to obtain a peptide of high D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-,~H- (CTOP) purity ( > 95%). Gel filtrations were performed on a I Sephadex G-15 (Pharmacia Fine Chemicals, PiscatD-Phc-Cys-Tyr-o-Tca-Orn-Thr-P~n-Thr-,,r~'Haway, N J ) column (2.65 x 75 cm), applying a 5% soI I D-Phe-Cys-Tyr-Q[ CH2N]-~-Tca-Orn-Thr-Pen-Thr-"H. lution of acetic acid isocratically, with a Buchler MonoD-Phc-C;s-Tyr-Q [CH,NH]-~-Trp-Orn-Thr-Pkn-Thr-h'H. static Pump (20-30 mL/h, over 20 h), a Buchler FractoScan (254nm), and a Buchler Automatic Fraction Collector. Preparative RP-HPLC was performed on a Perkin-Elmer Series 3B Liquid Chromatograph ~-Pgl-C;,s-+[ CH2N1-Tic-o-Trp-Om-Thr-Pkn-Thr-IZIH. equipped with an LC-75 Spectrophotometric Detector and an LCI-100 Laboratory Integrator, or on a Spectra-

-

Opioid peptides

I

"

\

gauche (-)

gauche (-)

gauche (+)

FIGURE 1 Conformational transformations of D-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (D-Tic). I. D-Tic with a free amino group prefers gauche( + ) side chain conformation. 11. Peptide chain attachment on N-tcrmini of D-Tic 1,2 pseudoequatorial strains (bend arrows) dcstabilizing the gauche( + ) conformation. 111. The heterocyclic ring flips to a gauche( - ) side chain conformation, in which both the N- and Csubstituents bear a 1.2 pseudoaxial relationship devoid of strains. IV. Sp3 hybridized N allows an out of plane relationship of both C- and N-termini, possibly allowing for gauche( + ) conformation. Note that guuche( + ) for a D-alnino acid corresponds to the same topography as gauche( - ) for an L-amino acid. TABLE 2 Analvtical data for peptides prepared in this work Peptide

1

2 3 4 5

6

Thin-layer chromatograph)" R, valucs

HPLCb

FAB-MS

k.'

I

I1

111

IV

0.32 0.29 0.29 0.3 1 0.26 0.25

0.79 0.78 0.77 0.78 0.76 0.80

0.77 0.76 0.75 0.75 0.76 0.78

0.76 0.73 0.73 0.72 0.73 0.74

11.45 7.25 6.47 8.36 7.66 6.12

[M + HI 10000 dimension; shifted sine-bell multiplication was applied 881 0 5 6.0 17 800 f 4400 in both dimensions prior to FT. Digital resolution in F1 91.2 5 11.9 5534 f 2014 was 5.2 Hz/pt, and in F2 was 2.6 Hz,'pt, in all cases. 9 10000 1439 f 215 Homonuclear dipolar correlated 2D NMR (NOESY) 9 10000 6419 f 104.7 was used to trace interresidual connectivities NH' I 891.9 f 72.7 s 10000 and CH:, as well as to provide important information 3.7 f 0.8 11532 116 regarding the secondarj structure of the peptide. +

406

Opioid peptides Much more dramatic effects of amide bond reductions on the p opioid receptor affinity and selectivity can be seen with peptides 2 (with p vs. 6 selectivity of 20.2), 5 and 6 (Table 3). An interesting observation comes from comparing the binding potencies of the amide reduced peptides 2, 3, 6 with their corresponding peptides 1, 111, 4, respectively (2). Though they are less potent, there is only a modest decrease in their selectivities for p vs. 6-opioid receptors. This suggests that while amide bond reduction decreased affinity for the p-opioid receptor, it does not contribute much to the discrimination between p and 6 opioid receptor types. The low affinity of 4 has been explained by a mismatched topography ofthat peptide, due to agauche( + ) side chain conformation of Tic', leading to an opposite arrangement of D-Phe' and Tic3 aromatic pharmacophores, this geometry not being recognized well by a p opioid receptor (3,23).

chain conformers. This result encouragingly showed that alkylated D- 1,2,3,4-tetrahydro-p-carbolinecan exhibit bias towards the g( + ) side chain conformation unlike the exclusive g( - ) observed for acjilated tetrahydroisoquinoline carboxylate (3). The proton spectral assignments and coupling con-

stants for D-Phe-Cis-Tyr-D-Tca-Orn-Thr-Pkn-ThrNH2 (1) are shown in Table 5 ; calculated side chain conformer populations and accessible $(NH-CH,) angles are given in Table 6. Several features of these data are noteworthy. First, quite unexpectedly, the acylated D-Tca4 residue has a side chain conformation biased towards a g( + ) conformation. Due to the intermediate values of the coupling constants (J,D) -2.5; 6.8 Hz)in peptide 1 it is postulated that a dynamic exchange between two conformational states, g( - ) and g( + ), takes place. At present the different behavior of D-Tic in peptides I11 and 3, and of D-Tca in 1 is not clear, since Conformational studies both amino acids are pipecolic acid analogues. A comIn order to establish the side chain preference of alky- parison of angles with those of I (21) and I1 (1) lated 1,2,3,4-tetrahydro-/3-carboline,conformational suggests that they are still compatible with a type 11' p studies were performed on the simple model compound, conformation (Table 6). Similarly to I, the side chain Boc-Tyr(O-2,6-Cl~-Bz1)-~[CH~N]-~-Tca-OMe, the conformation of peptide 1 is biased towards g( - ) for synthesis of which additionally allowed us to establish D-Phel, but there is a high population of a trans rotamer the best conditions for the desired series of reductive of Cys' (t and g( - ) for 1). Also, the side chains of the alkylation reactions on the resin. Spectral assignments amino acid preceding and following D-Tca (Tyr3 and were obtained on the basis of a phase sensitive COSY Om5, respectively) exhibit some conformational disorexperiment, and are tabulated in Table4. Because of der in comparison with I(21). While the side chain of considerable spectral overlap of CH,/CH2 signals of Orn' in 1 seems to be almost equally divided among all D-Tca, detailed spectral decoupling experiments were three staggered states (mostly g( - ) for Lys5 in I), Tyr' needed. The nonequivalence of both vicinal coupling has a preference for a trans (g( - ) in I) side chain constants and the relatively low value of J,p suggest a Conformation. dynamic equilibrium between the g( + ) and g( - ) side Comparing 1 with I there is an upfield shift of the Cys: ( -0.32 ppm) and amide protons ( -0.13),a downfield shift of the Tyri (0.44 ppm), as well as a dramatic downfield shift of Orn: (0.80 ppm with respect to Lys5 TABLE 4 Chemicnl shjr und coupling constant ussignnients for model cornpound of I) protons. As has been observed for other peptides with constrained amino acids, the ring current anisoFH6J-DMSO, N"-Boc-T~r~0-2,6-C12-Bzl)-~[C~~NJ-D-TcnOMe, tropy of the D-Tca4 residue seems to be responsible for 303 K these effects. Nuclear Overhauser effects are in good agreement with the above analysis. There is a very Residue Chem. shift [ppm] J [Hzl strong cross-relaxation signal for alpha protons of Cys' =Y r and Pen', attributable to a negative disulfide helicity But 1.29 (s) (24). Additionally, the characteristic CH;/NH' cross6.67 a/NH = 8.7 NH relaxation signals are observed for the following pairs r 3.80 (m) of amino acids: 112, 415, 611, 718. Thus these parts of 8' 2.84 (dd) alp' = 4.4 B'/8" = 13.9 amino acids are linked via trans peptide bonds (25). No B" 2.55 (d4 x/B" = 8.9 Cys:/Tyr3NH (2/3) or Orn5CH,/Thr6NH (5/6)crossrelaxations have been observed, but a significant cross(d) CH, 2.75 relaxation effect was detected for Orn5NH/Thr6NH.In D-Tca light of these results, and even with the presence of a i( (dd) ./B' = 3.3 conformationally constrained D-Tca residue in the core a/P" = 5.9 of the turn, a type 11' p turn is the best description of 8 2.99 (m) the backbone conformation of peptide 1. This is supN-CH2 4.07 (d 1 NCH/NCH = 14.8 ported by the very low temperature coefficient of 3.93 (d) Thr6NH, suggesting that the amide is either hydrogen (S) OMe 3.53 bound (to the Tyr3 carbonyl group) or solvent shielded. $J

+

407

W.M. Kazmierski e t a / . TABLE 5 Chemical shifrs ( ~ H Jcoupling , wisfanr a~iigrinierirs,arid aniide reniperariire coeficients for peptide.7 u-Phe-C~:s-T~r-D-Tca-Om-~hv-P~n-Thr-

NH,, 1 . arid o-Phe-C;,s-Tic-o-Trp-Orn-Thr-Ph-Thr-NH,. 4,/-'HI,-DMSO, 303'K Residue

Chcmical shift [ppm] Tciiip. factor [ 10 'ppm:deg]

Coupling constants [ H z ]

~

D-Phe' NH; A z

P' B" Cys' NH A 2

B' P" Tyr' 1 Tic' 4 NH A x

1

4

8.07 (rn) - 1.04 3.20 (rn) 3.29 3.00

8.06 (m) - 0.44 4.02 3.23 1.96

9 '71 -5.1 5.32 3.05 2.80

N-CHz

-

2

N-CHZ

om5 NH A 2

8'

8" 7

6

2

B i

OH

408

P')B'' = 14.0

4.8 9.5 13.8

r / N H = 10.2

8.7

~ p = '5.9 D" = 10.1 8'1"' = 15.5

4.6 10.0 13.6

= 9.9

2

r/NH 5.22 2.95 2 84 4.89 (m)

8.09 4.7 4.27 3.04 2.96

5 25 3 07 2 93 4.70 J 50 (dd)

=

7.9

218' =

7.0

8.4 - 7 7

-

4.00 1.92 1.54 149 2.65

7 40 - 0 10 4 43 4 03 I .03 -1 8

z/b"

B'iB''

= 8.5 =

13.2

4.1 5.8 15.8

-

6.2 ZIP' =

2.5

2,'P'' = 6.8

P'/D''

=

16.5

6.6 8.8 14.2

14.8

6-NH; Thr6 NH A

zip' = 5.2 xlp"

-4

8.59 5 03 >.99 2.50

B' P"

4

- 4.0

B' B" D-Tca' 1 D-Trp4 4 NH A

9.25 08 5.47 3.20 2.92

1

8.29 3.1 4.07 1.70 1.25 1.18 2.58 7.63

7.28 0.84 4.39 3.88 0.91 -4.9

z:NH

= 7.5

8.0

218'

= 4.8

218"

= 6.4

4.7 9.1

r / N H = 7.4

7.8

zip= 7.4

4.5 6.3

-

lr'i6= 6.3

Opioid peptides TABLE 5 (continued) ~

Residue

Pen’ NH A 2

i

Chemical shift [ppm] Temp. factor [ 10-3ppm/deg]

Coupling constants [Hz]

1

1

4

8.27

- 6.3

2,”H

7.92

4

= 9.4

9.0

r/NH = 9.7

8.3

@= 3.6 fill.= 6.3

3.9 6.4

- 2.0

4.88

4.80

1.2411.28 0.041

1.27/1.40 0.13 1

Thrn NH A

8.31 - 4.1

2

4.30

B

3.98 1.06 x5.01

Y OH

8.32

- 4.4

In summary, substitution of D-Trp4 by D-Tcal does not alter the peptide backbone conformation. The constrained nature of this cyclic amino acid, and its apparent ability to flip between both gauche( - ) and gauche( + ) conformational states, perturbs the side chain conformation of the neighbouring residues: Cys2, Tyr3, and O r d . Another interesting observation from the NOESY experiments is a strong cross-correlation between the Tyr’CH, and both N-CH2 protons of DTca4. The chemical shifts, coupling constants, and temper-

4.26 4.01 1.05 4.90

pseudopeptide bond are in intermediate exchange at 303 OK, and as such are observed as two broad humps. This observation may be related to a two-site exchange between the gauche( - ) and gauche( + ) conformations of the D-Tca4 side chain. The 4 dihedral angles between TABLE 6 Side chain conforiner population and accessible @ angles derived,froru

‘ H NMR artalpis of D-Phe-C?k7)r-o-Tca-Orn-Thr-Ph-Tlrr-NH2, 1, f-’Hri]-DMSO.303 K

ature coefficients of ~-Phe-Cys-Tyr-flCHzN]-~-TcaI

Om-Thr-Pen-Thr-NH2, 2, are listed in Table 7. Due to the low resolution of some signals in the 1D spectrum (its possible origin will be discussed later) at 303 O K , we decided to examine the molecule at 365 K. However, the 2D experiments (low spectral resolution is acceptable) were carried out at 303 ’K. Interestingly, peptide 2 consists of two domains, a flexible one (amino acids 2-4) and a rigid one (amino acids 6-8). Coupling constants could be measured for the semi-rigid domain amino acids, but not for the flexible ones. Differential decoupling experiments failed to extract the vicinal coupling constants for amino acids directly connected with the flexible peptide bond isostere (Cys’, Tyr3, D-Tca4, Om5). Visible broadening of amide and alpha proton signals for these residues may be attributed to conformational averaging. Signals of residues not in the “flexibility domain” (Thr6, Pen7, Thrs) had sharp resonances. Comparison of the 1D spectra of 2 at 303°K and 365 OK besides improving spectral resolution (due to faster molecular tumbling) revealed some interesting dynamic processes. The diastereotopic protons of the niethylene group adjacent to the amino group in the

$ I angles

Residue

Rotarner population ( ” ” )

0 28.6 71.3 120

-

0 83.5 16.5 -

156,

-

87, 60

0 64.1 35.3

*

-

160.

- 82,

38, 82 39.5 39.6 20.9

-

162, -

-

144,

-

80, 36, 84

-

96. 60

-

100

-

-

150,

-

409

W.M. Kazmierski et a/. TABLE 7 Chemical shifrs, coupling constunrs and crnride rerirperariire coeficienrs .for o - P h e - C ~ , . s - T , . r - ~ [ C H ~ ~Trp-Om~ H l - ~ -Tlir-Ph-Thr-NH2(3). o-Plie-C~s-T~r-~[CH~N~-o-Tca-Orn-Tlir-P~riThr-NH 2 (21.u-Pgl-Ci'~s-~/CH~~V/-Tic-n-Trp-Onr-Thr-P~,~-Tlir-NH~ (5). in 12Hn]DMS0 Chernictil shifi [ppin J A . Tenrp. jictor 10 '[pph:deg/

Residue

Coiipl. c'onsiani [Hi]

~

3 333 K

2 365 K

333 K

8.04 4.13 3.23 2.97

8.21 4.19 3.23 2.95

8.63 5.26 -

8.80

7.67" ND 3.60 -3.12 s 3.08 2.77

D-Pgl' 5 D-Phe' 3 NH x

8' 8" Cys' NH A

9.02 - 4.6

-

CH? 5

Tyr3 Tic' 5 NH x

B' B" CH: 3,2 D-Tca4 2 D-T$ 3 3 NH r

P' P" Om5 NH A

-

8.15 5.5 4.17 2.87 2.61 ND

ND 4.0 I 3.23 3.34

-

x

B' 8" 8 8-N;

Thr6 NH A r

B 7 OH

Pen' NH A x .,' i

",,I

4 10

4.80 3.15 2.95 -

4.86 3.23 2.97

x

8' B"

A

- 3.5

8.56 1.7 3.92 1.32d 1.02'' 0.98* 2.44d 7.70

7.28 1.8 4,31 3.93 0.98 z 5.3" -

-

8.15 2.4 4.69 1.31 1.37

2

3

5

zip'

= 4.5

rip''

= 9.4

B'iB"

=

14.3

ziNH

=

8.9

x,B' = 3.5 r;B" = 9.4 8'8" = 14.3

5

5.1 9.1 14.1 8.3

NDb

ND ND ND

ND ND ND

7.87 5.7 4.16 2.72 2.66 2.8 1

3.88"

-

4.18 ND 2.96

8.44 4.65 3.16 3.04

r/NH = ND rip' = ND zip" = 8.7 B','B" = 13.5

ND 9.1

s

7.8 6.2 8.2 16.6

8.20 1.4 4.25 1.85 1.61 1.61 2.81 7.77d

8.44 ND 4.12 1.75 1.38 1.32 2.67 7.70

r/NH = 7.8

8.0

7.8

z/B'-ND x ' /8"-N D

ND ND

ND ND

7.57 3.8 4.35 4.00 I .06'

7.32

r/NH = 7 . 6

7.2

3.9

4.3 1

ZIP=

4.5

3.94 0.98

8:;l = 6.3

5.3 5.9

6.1 6.3

8.04

7.78

xjNH = 9.1

9.0

6.9

ND

ND

-

-

-

2.82 2.75

x/NH = niL

7.6

= 5.7

ND ND ND

zip'

x / 8 " = 9.7

B','"'

=

14.3

ND ND ND

-

-

- 4.5

4.64 1.32 1.38

4.51 1.19 1.22

Opioid peptides TABLE 7 (continued) Residue

Chemical shift [ppm] A, Temp. factor 10-3[ppb/deg]

3 333 K Thr* NH A

-

r

B I

OH a

7.90 1.6 4.26 4.00 1.05 4.98d

2 365 K

-

7.80 3.4 4.24 4.00 1.08"

Coupl. constant [Hz]

5 333 K

3

2

5

7.96

r / N H = 9.1

8.5

8.6

4.20 4.01 1.06

a/B= 3.9

3.7 6.3

3.8 6.3

6.3

Probable assignment. ND could not be determined, see text. Multiplet. 303'K. Assignments could be reversed (Thr6/ThrM).

NHi-CH1crare compatible with either a type 11' P turn or a reverse y turn conformation. Analysis of the NOESY spectra reveals that two important cross-peaks between D-Phe'a/Cys2NH and Cys2a/Tyr CHiNH were not present. These amino acids are part of the flexible domain in 2. It is well known that internal flexibility in molecules greatly influences the magnitude of NOE cross-peaks. Nonetheless, the presence of a 4/5 and the absence of a 5/6 CH:/NHi+ NOE can be interpreted as evidence for a type 11' P-turn conformation involving residues 3-6. However, protons of Om5 and Thr6 was missing. Moreover, analysis of amide temperatures factors did not indicate H-bond formation by the Thr6 NH. On the other hand, the low temperature factor for the Om5 amide NH may be indicative of a y- or inverse y-turn involving residues 3-5 (26). Analysis of the amide chemical shifts for compounds 1 , 2 and 3 (Table 5 and Table 7, respectively) reveals no essential differences. The major discrepancy, an upfield shift of the Tyr2 CH2 amide proton of 2 (-0.28 pprn relative to 3) is expected as a result of the peptide bond replacement. The same, to a lesser extent, is true for the Cys' amide proton ( -0.22 ppm upfield shift relative to 3 ) . Otherwise, the chemical shifts for other residues are very uniform (with a small exception for the Thr8 amide). I

I

D-Phe-Cys-Tyr-IC/ICH~NH]-DTrp-Om-Thr-Pen-ThrNH2, 3, is a model compound with a single peptide bond isostere modification, designed to test whether there is any significant influence of CH2/C=O substitution on peptide conformation before considering more complex cases involving cyclic amino acids such as Tic

or D-Tca. Since chemical shift anisotropy is an important tool in conformational analysis, it was of considerable interest to establish the magnitude of influence of this structural modification on the chemical shifts of the neighboring nuclei. There are several features that can be noticed immediately in the IH NMR spectrum of 3. First, it was possible to obtain only limited information about vicinal coupling constants for ~ - T r pand ~ , the signal for the ~ - T r NH p ~ was not found. Additionally, the Tyr3 amide appeared as a broad singlet instead of the expected doublet. These irregularities in the 'H NMR spectrum of 3 were limited to the 3rd and 4th residues only. The aromatic moiety in the Tyr3 residue showed particularly interesting properties. Instead of the commonly seen pair of complex doublets of an AA'XX' system of Tyr (2', 6' and 3',5' going upfield, respectively), a split signal (two doublets of uneven intensity) for the 2', 6' protons were found at 303 OK (Fig. 3). With an increase in temperature both signals started to fuse and at 333 "K they coalesced into a doublet. An estimation of the activation enthalpy for the two site jump of the tyrosyl ring gives about 77.3 kJ/mol. This finding represents a rare experimental demonstration of restricted rotation of an aromatic side chain in a small peptide, which is slow enough to be observed on the NMR time scale (less than lo3 s - I ) (27). Chemical shifts for 3 in Table 7 are strikingly different than these for I(24). The expected upfield shifts (as a result of isosteric peptide bond replacement) of the Cys2NH ( -0.32 ppm) and the Tyr3 N H ( -0.46 ppm) are easily identified. Other significant upfield shifts include the ThrhNH ( -0.41 ppm), Pen' NH ( -0.34 ppm) and Thrs NH ( -0.49 pprn), whereas a downfield shift is observed for the Oms amide proton (0.32 ppm). Sim411

W.M. Kazmierski et al.

NOE cross-relaxation signals for the D-Trp:/ Orn'NH and OrnsNH/Thr6NH pairs of protons are diagnostic for type 11' p-turns. There also is crossrelaxation between the alpha protons of Cys2 and Pen7, and unexpectedly, cross-relaxation between Cysi and Pen: protons. Other NOE cross-peaks, expected if the peptide bond is trcms, also are found: CH:/NH'+ I , for i and i + 1 in residue pairs 1/2, 2/3, 6/7, and 7/8, respectively. A type 11' turn precludes the spatial proximity of Orn5CH, and Thr6 NH, and indeed, cross relaxation from these residues is not detected. Side chain population analysis is incomplete because it is not possible to extract the appropriate vicinal coupling constants, presumably due to the flexibility. However, some important observations can be made. For example, there is a large tram side chain population of Cys' (90"". compared with the almost equal distribution among irons and gauche( - ) states for I). Also, the side chain of the Tyr' CH. is mostly trans populated (gcruche( - ) for Tyr in I). This effect may cause the observed chemical shift variations bctween equivalents residues of I and peptide 3. The low value of the Thr6 vicinal coupling constant J,o (4.5 Hz) vs. 6.8 Hz for CTP is another discrepancy between these two peptides. In summary, peptide 3 still may be best characterized by a type 11' 8-turn conformation of its backbone involving residues 3 to 6, though there is substantial flexibility of its backbone, best manifested by a slow twosite jump of the Tyr3 ring. The chemical shifts, coupling constants, and temperature data for D-Phe-CGs-Tic-D-Trp-Om-Thr-Pkn-ThrNH? (4), coefficients are listed in Table 5. KarplusBystrov (28) analysis of accessible @ angles does not indicate any major difference with those of I (24). Analysis of the coupling constants for Tic3 indicates that r I 1 i 1 ~ 1 1 1 v ~ i both vicinal coupling constants are almost equal as a result of the somewhat skewed gauche( + ) conforma7.20 7.10 7.00 6.90 6.80 6.70 6.60 tion of the Tic3 side chain group (Table 5). Similar to FIGURE 3 peptide 3, one observes an increased trans side chain I population of Cys' (probably the result of repulsions Temperature dependence of aromatic resonances in D-Phe-Cj s-T!rfrom the Tic3 aromatic ring). This allows only moder+[ CH2NH]-o-Trp-Om-Thr-P&-Thr-NH-. 3. ['H,]-DMSO. ate cross-relaxation between the alpha protons of Cys2 and Pen7. On the other hand, NOESY experiments reveal two important cross peaks: Cys2CH,/Tic3 NCH2 ilarly, for alpha protons, there are dramatic upfield shifts and Tic3 CH,/Tic3 NCH?. The first effect can be obof Cys' (-0.81 ppm), Pen7 (-0.32ppm). and Tyr' served only if the corresponding peptide bond is in a (-0.42 ppm), all a consequence of the peptide bond trans configuration (25). The 1D spectrum of 4 suggests replacement. Some of the unexpected shifts (e.g. for that only one conformer is observed. Thus, in contrast Pen7) may be related to the oscillation of the tyrosine to the proline ring where cis and trans peptide bond aromatic ring between two sites and a transannular isomers are often in equilibrium, the tetrahydroisoquinoline ring seems to exclusively prefer a trans conforanisotropic effect. Analysis of the NH-CH, dihedral angles (obtained mation of the peptide bond. NOESY experiments refrom Table 7) supports the possibility of a type 11' 8- veal that the following pairs of protons CH',/NH'+ are turn of the backbone, though the temperature coeffi- in close proximity: 1/2, 3/5,4/5, 6/7, 7/8. These peptide cient of Thr6 NH is not as small as usually observed in bonds are tram. Lack of a 5 i 6 cross-relaxation peak in H-bonded amides. On the other hand, the observed the presence of a strong NH5/NH6 signal again is in-

'

412

Opioid peptides Psi ($) angles calculated from the vicinal coupling constants for ~ - T r and p ~ Thr6 exhibit a significant deviation compared with the corresponding values in I (24). NOESY experiments did not add any additional information. Only intraresidue NOE signals were deNtected. H Thus, the conformational data attained was inconclusive for this peptide. The above analysis clearly shows that reduction of peptide bonds in cyclic constrained peptides with an otherwise rather semi rigid backbone (23,29), can result in a greater degree of conformational flexibility. In some specific cases, a slow interconversion between two states (as in peptide 3) may be observed. These observations are supported by the complementary results of Marraud etal. (29) who found different conformations for protonated vs. non-protonated forms of identified for ~-Pgl-C~!s-flCH2N]-Tic-~-Trp-Ornreduced amide bond peptides. I Thr-Pen-Thr-NH2 (S), in the form of broad lines for some signals at 303 O K . Increased resolution was CONCLUSIONS achieved by doing NMR experiments at 333 OK. Chemical shifts and coupling constants obtained are listed in We have demonstrated that peptides with secondary Table 7. However, even at elevated temperatures, the reduced peptide bonds can be easily constructed in a signals of the 2nd, 3rd, and 7th residues still remained reductive alkylation reaction on a solid support as has sufficiently broadened that no accurate coupling con- been previously done for peptides with primary peptide stant information could be measured. To obtain qual- bonds (8). There is an increasing interest in this class itative information about the conformation and dynam- of peptide amide bond modified peptides, since several ics of this molecule, chemical shift analysis (in a similar of them possess antagonistic (30) or inhibitory (31) manner as done before) was utilized. Considering the properties. In agreement with our earlier observations (3, 28), alpha protons first, there was a significant upfield shift of the Cys2CH, protons in 5 (-0.87 ppm compared to acylated Tic residues prefer a gauche( + ) side chain 4). This is much greater than one would expect solely conformation, as was found for peptide 4 in this series. as a result of isosteric peptide bond replacement with In contrast, in the closely related cyclic D-tryptophan an alkyl amino group (about -0.4 pprn). In addition, - i.e. D-Tca-containing peptide 1, there seems to bc a there was a tremendous upfield shift of the Cys2NH bias towards a gauche( - ) side chain conformer, with resonance in 5 relative to 4 ( -1.58 pprn). These results possible dynamic equilibrium between the two states strongly implicate dramatic ring current anisotropic ef- (gauche( - ) and gauche( + )). Both Tic and D-Tca fafects, and point to very different orientations of the Tic vored trans peptide bonds unlike some other amino aromatic side chains in 5 and 4. Both the alpha and acids (Pro, etc.). We attempted to stabilize the amide protons of ~ - T r in p ~5 experience a downfield gauche( - ) conformation of Tic and the gauche ( + ) shift relative to 4 (0.38 and 0.35 ppm, respectively). A conformation of D-Tca by synthesizing peptides with closer look at other alpha and amide protons of pep- flCHzN]Tic (5,6 ) and flCH2N] D-Tca (2) reduced tides 4 and 5 (Tables 5 and 7) reveals that with the peptide bonds. This would decrease 1,2 diequatorial exception of the alpha protons of Pen7, most of the repulsions (Fig. 1. 11) as a result of thc presence of corresponding resonances are not very different. Fi- tetrahedral nitrogens (Fig. 1,lV). Interestingly, peptides nally, the alpha proton of Tic in 5 experiences an upfield 2, 3, 5 and 6 exhibited a significant degree of conforshift of - 1.34 ppm, which is much larger than expected mational mobility (on the NMR time scale) in the vifrom an alkyl amine peptide bond modification. Again, cinity of the reduced peptide bonds, thus resulting in anisotropic effects of the aromatic ring of Tic3 must be broadening of resonances for amino acids adjacent to involved. the reduced peptide bonds. As a result, it was not alAnalysis of a 1D spectrum of 5 revealed that while ways possible to obtain sufficient NOE and J-coupling D-Trp4, Om5 and Thr6 gave reasonably sharp signals, data for comprehensive conformational analysis of the next amino acid Pen7 again gave broad (slow ex- these peptides. However, amino acid residues away change) alpha and amine proton resonances. Most from the peptide bond modification showed characterconvincing, however, is a comparison of the chemical istics of a relatively stable backbone conformation shift difference between the diastereotopic methyl (sharp resonances, significant NOES, etc.). The recepgroups of Pen7. This value is only 0.033 pprn for 5, tor binding data (Table 3), demonstrate fairly compacompared to 0.13 1 ppm for the more rigid 4 (Tables 5 rable binding potencies to the p opioid receptor for analogues 1 and 3 as well as for 4 and 6.Thus, despite and 7). terpreted as evidence for a type 11' p-turn backbone conform ation. The chemical shift difference between the diastereotopic methyl groups of Pen7 is much larger in 4 (0.132 ppm) than in I (0.06 ppm). Table 5 reveals that there is a substantial upfield shift of the D - T ~ ~ ~ (-0.74 ppm in comparison to I), which possibly is a result of ring anisotropy from the gauche( + ) populated side chain of Tic, in contrast to the mostly gauche( - ) populated side chain of Tyr in CTP, I. Upfield shifts of the Thr6, Pen7, and Thr8 amide resonances, and the absence of this phenomenon for Orn5NH, indicates the presence of transannular ring current anisotropy effects on these residues. Strong indications of conformational averaging were

413

W.M. Kazmierski et al. the findings from NMR studies that the reduced peptide bond analogues are substantially more flexible than their closely related analogues with standard peptide bonds, they apparently are still able to attain receptor bound conformations, similar to those of their respective parent compounds. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service Grant NS 19972, DA 04248 and DA 06284. The mash spectral determinations were performed by the Midwest Center for Mass Spectrometr) at the University of Nebraska. a National Science Foundation Regional Instrumentation Facility (Grant No. CHE 811 1164).

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J.E., Burks,T.F.,Yamamura. H.I.& Hn1by.V.J. (1988)J. Med. CheiJr. 31. 2170-2177 Kazmierski, W.M.. Yamaniura. H.I. & Hruby, V.J. (1991) J . A m . Cheni. Soc. 113. 2275-2283 Sugg. E.E., Griffin, J.F. & Portoghese. P.S. (198S)J. Org. Chenr. 50, 5032-5037 Toniolo, C.. Bardi, R., Piazzesi, A.H.. Crisma. M.. Balaram, P.. Sukumor, M. & Paul, P.K.C. (1989) In Peprides 1988 (Jung. G. & Bayer, E., eds.). pp. 477-479, W. deGru)Ter. Berlin Johnson, F. (1968) Chem. Rev. 68, 375-413 Rodriguez, M., Ball, J.-P.. Magous. R.. Castro, B. & Martinez, J. (1986) Int. J . Peptide Prorein Res. 27. 293-299 Coy, D.H.. Hocart, S.J. & Sasaki, Y. (1988) Tetruhedron 44, 835-841 Spatola. A.F. (1983) Cheinisrrj ctnd Biochewi.frrr u/Anrino Acid.?. Peptides and Proteins (Weinstein, B., ed.) pp. 267-357. Marcel Dekker, New York Upson, D.A. & Hruby. V.J. (1976)J. Org. Cheni. 41, 1353-1358 Stewart, J.M. & Young, J.D. (1984) SolidPhnw Peptide Swrhesis. 2nd edn., Pierce Chem. Co., Rockford. IL Orlowski, R.C.. Walter, R. & Winkler, D. (1976)J Org Cherv.. 41, 3701-3705 Kaiser, E.. Colescott, R.L., Bossinger, C.D. & Cook, P.I. ( 1 970) 34. 595-598 Christensen, T. (1979) Peprrdes. Strurritre and Firncriort. (Gross, E. & Meienhofer, J., eds.). pp. 385-388, Pierce Chem. Co.. Rockford, IL

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15. Harvey. D.G. (1941)J. Chem. Soc., 153-159 16. Fehrentz. J.A. & Castro, B. (1983) Synthesis, 676-678 17. Fisher, L.E. & Muchowski. J.M. (1989) Org. Prep. Proced. Int. 22, 399-484 18. Hocart. S.J., Murphy, W..4. &Coy. D.H. (1990) J . Med. Chem. 33. 1954- 1958 19. Schbvartz, H.. Bumpus, F.M. & Page, I.H. (1957)J. Am. Cherir. SOC.79. 5697-5703 20. Marion. D. & Wiithrich, K. (1983) Biochern. Biophys. Re.r cO l7 tlJ lU tl. 113, 967-974 21. Rance. M., Sorensen, O.W., Bodenhausen, G., Wagner, G., Ernst, R.R. & Wiithrich, K. (1983) Biochem. Bioplrys. Res. Cwnmun. 117. 471-478 22. Bax. A. & Freeman, R. (1981) J . Mag. Resonance 44. 542-561 23. Kazmierski, W.. Yamaniura. H.I., Burks, T.F. & Hruby, V.J. (1989) in Peptides 1Y88 (Jung, G. & Bayer, E., eds.), pp. 643645. W. deGruyter, Berlin 24. Sugg, E.E., Tourwe, D.. Kazmierski, W., Hruby, V.J. & Van Binst, G. (1988) In!. J . Pepride Protein Res. 31, 192-200 25. Wiithrich. K., Billeter, M . & Brown, W. (1984) J . Mo1. Biol. 180, 7 15-740 26. Smith. J.A. & Pease. L.G. (1980) Crit. Rev. Biochem. 8. 315-399 27. Ronianowska, K. & Kopple, K.D. (1987) Int. J . Pepiide Protein Res. 30, 289-198 28. Bystrov. V . F . (1976) Pro,q. NMR Spectroscopj, 10. 41-81 29. El Masdouri, L., Aubry, A,, Sakarellos, C., Gomez, E.J., Cung, M.T. & Marraud, M. (1988) Int. J . Peptide Protein Res. 31, 425-428 30. Hocart. S.J.. Murphy, W.A. & Coy, D.H. (1990) in Peptides. Chemistry. Srntcture and Biologj (Rivier, J.E. & Marshall, G.R., eds.), pp. 233-23.5, ESCOM, Leiden 31. Toth, M.V.. Chiu. F.. Glover. G.. Kent, S.B.H., Ratner, L., Van der Heyden, N., Green, J., Rich. D.H. & Marshall, G.R. (1990) in Peptide.?, Cheiiiisiry, Sfructure and Biology (Rivier, J.E. & Marshall. G.R.. eds.), pp. 835-838. ESCOM, Leiden

Address: Dr. L'rctor J . Hruhj Regents Professor Department of Chemistry University of Arizona Tucson, AZ 85721 USA