COMMUNICATIONS TO THE EDITOR
Dec. 20, 1963
Bz
BUO.NHNHz
BUO.NHN=C (CHs)2
!
IV
I
8' 1 ?Bz
H..Pt
j.
.'
BU0,NHNHCH (CH3)z V
J.
CHz
1
COCl*
1x1
O C N ~ H C O ~ O CI ~ H ~
R
Bz I
I
1 HCI
BI-0 SHSCO-T~~.OCZH~ 2 . NaHCOa 1-1 R Bz CBO I I HzSSCO-Tyr OC2H, 1-111 NO2 R BZ
-___f
Arg(NOz) O H
+
DCCI
I
CBO . Arg-NHdCO-dyr. IX
NaOH
OCzHs
--+
NO*
R
I
1
C B 0---Arg-NH
NCO-Tyr
x
BUO
. OH
(CH1)aCOCO-;
=
CBO
=
OCHtOCO-
;
Bz
=O
-C
O - ;
H3C UCCI
=
S,S'-dicyclohexylcarbodiimide; R
=
\
/cHHac (3 : 1 : 3 : l), m.p.
methanol-water-chloroform-benzene 1-k---146', [ a I z 4 D - 4 3 O , Xmax 271 mg ( e 16,850) ( A E d . Calcd. for C63H70NI4O13: C, 57.29; H , 6.32; N, 17.65. Found: C, 57.60; H , 6.50; N , 17.44). Cleavage of the benzyloxycarbonyl protecting group of XI with hydrobromic acid in acetic acid and subsequent base treatment yielded N- [2-isopropyl-3-(nitro~ - a r g i n y l-carbazoyl]-L) tyrosyl-~-valyl-Lhistidyl-Lprolyl-L-phenylalanine methyl ester (XII), m.p. 1241:30°, [ a ] ~ -57' (ilnal. Calcd. for Ci6H64N140~1: C, , 5 3 1 ; H, 6.60; N,2 0 . 0 i . Found: C, 55.32; H, 6.39; N , 19.50), which was condensed with benzyloxycarbonyl L-aspartic acid-@-benzylester13 under the influence of dicyclohexylcarbodiimide to afford N- [2-isopropyl - 3 - (benzyloxycarbonyl - [ [@-benzyl] ] - L - aspartylnitro - L - arginy1)carbazoyll- L- tyrosyl- L-valyl - L - histidyl-L-prolyl-L-phenylalanine methyl ester (XIII), m.p. 136-1452', [o]"D -40' ( 4 E U l . Calcd. for C64&3N1501~.H20:C, 57.61; H, 6.27; N , 15.75. Found: C, 57.29; H, 6.35; N, 15.56). Scission of the benzyloxycarbonyl, benzyl ester, and nitro groups of XI11 by catalytic hydrogenation and then treatment with concentrated hydrochloric acid a t 40' for 1 hr. to remove the methyl ester f ~ n c t i o n 'provided ~ the free isosteric octapeptide ( I ) . Purification was achieved by countercurrent distribution in the systems n-butyl alcohol-water and sec-butyl alcohol-water to give I as an amorphous solid, m.p. 193-19S0, [ a ] D Z 3 -33' (water). Homogeneity was established by paper electroph~resis'~ ( 1 3 ) Cyclo Chemical Corp. (14) R . B. Merriiield and D. W. Woolley, J . A m . Chem. Soc., 7 8 , 4646 (1956). (15) A hlisco paper electrophoresis apparatus and organic buffers containing 10Vc urea were used for these experiments as described by I,, N . Werum, H. T Gordon, and W. Thornburg, J . Chvomafog.,3 , 125 (1960).
404 1
(single spot with K3Fe(CN)6-FeC13 a t pH 4, 7.2, and 8) and paper chromatography16(Rr (1) 0.38; Rr ( 2 ) 0.30; Rt (3) 0.45; single spot with K3Fe(CN)~-Fecloand p-nitrobenzene diazonium fluoroborate and Sakaguchi reagents). Quantitative amino acid determination gave the following molar ratio: Asp, 1.1; Arg, 0.9; Tyr, 0.9; Val, 1.0; His, 1.0; Phe, 1.0; proline was not determined.l 2 Biological activity was evaluated on the isolated rat uterus and through blood pressure measurerents in intact, phenobarbital-acesthetized rats. Isostere I has '/100th to ' / 2 0 0 t h of the activity of Val5-angiotensin 11-Asp1-@-amide(XIV) l7 in these assays and produces a twofold increase in' duration of pressor action over XIV in the rat a t doses which give an equivalent absolute response. The corresponding isosteric C-terminal hexa- and heptapeptides, synthesized by similar methods, exhibited 0.2% and 50-100%, respectively, of the activity of I. Structure-activity studies to date have indicated that, in order to be active, analogs of angiotensin 11 must contain the pentapeptide sequence Tyr-Val (or 1leu)-His-Pro-Pheplus a t least one additional amino acid attached a t the N-terminus. The present results show t h i t peptides in which this amino acid has been replaced with -NHN(R)CO- retain significant biological activity. This suggests that, even in the interior of a peptide chain, the isosteric moiety is able to assume a conformation which resembles that of an amino acid. The implications of isosteric replacement of amino acids in a peptide chain to such problems as susceptibility to enzymatic degradation will be the subject of a subsequent publication. Acknowledgment.-We are grateful to Dr. J. W. Constantine of our Pharmacology Department for the biological determinations. (16) The R i values (on Whatman paper N o. 4) refer t o the following ( 1 ) sec-butyl alcohol-formic acid (88%)paper chromatographic systems: water ( 7 : 1 : 2 ) , (2)ethyl acetate-pyridine-water ( 1 2 : 5 : 4 ) ; ( 3 ) methyl isobutyl ketone-formic acid (W%)-water (2: 1: 1). (17) Hypertensin-CihaB. This material produced a n average increase of 50 mm. in r a t blood pressure following intravenous administration of 0.1-02 pg./kg. Cf. F. Gross a n d H. Turrian in "Polypeptides Which Affect Smooth Muscles and Rlood Vessels," M. Schacbter, E d . , Pergamon Press, New York, N. Y . , 1960, p. 137.
MEDICALRESEARCH LABORATORIES
H A N S - J ~ ~ R G EHESS X WALTERT. MORELAND GROTON, CONNECTICUT GERALD D. LAUBACH RECEIVED OCTOBER 2, 1963 CHAS. PFIZER AND C O . ,
INC.
Geminal Proton-Proton Coupling Constants in CHz=N- Systems'
Sir : I t is commonly known2 that J"(gem) in the sp2type CH2 groups of olefins is usually small in magnitude and can be either positive or negative; there is a fairly good inverse correlationzh with the electronegativity (Ex) of the substituent in CH2=CH-X compounds. These olefinic J"(gem) values fall out(1) Part 111 of t h e series " N M R Spectral Studies of sp2-type CHz Systems." For Part 11, see B L Shapiro, K M Kopchik, and S. J . Ebersole, J Chem. P h y , . , in press ( 2 ) E . g . (a) C. N Banwell, A. D. Cohen, N . Sheppard, and J . J Turner Proc. Chem SOL., 266 (1959); (b) C . N . Banwell and N. Sheppard, Mol P h y s . , 3 , 351 (19601, (c) C . N Banwell, N. Sheppard, and J . J . Turner, Specfrochim A c f a , 16, 794 (1960); (d) E B Whipple, J H . Goldsttin, and L. Mandell, J . A m . Chem. Soc., 81, 3010 (19601, (e) W. Bruegel, T h . Ankel, and F . Krueckeberg, I: Eleklrochem., 64, 1121 (1960); (f) A A B o t h e r - B y and C . Naar-Colin, J . A m . Chem s o t . , 8 3 , 231 (1961), (g) G S Reddy, J . H Goldstein, and L. hlandell, i b i d , 83, 1300 (1961); (h) T Schaefer, C a n . J Chetw.. 40, 1 (1962); (i) A . A . Bothner-By, C. .L'aar-Colin, and H . Giinther, J . A m Chem. SOL.,84, 2748 (1962J, (j) G S. R e d d r and J. H . Goldstein, J M o l . SPectry , 8 , 47.5 (1962), (k) R . T . Hobgood, Jr., G S. Reddy, and J H Goldstein, J . P h y s . C h e m , , 67, 110 (1963).
4042
COMMUNICATIONS TO THE EDITOR
VOl. 85
TABLE I Compoundo
JIaa(gem)’ (c P s
CHz=N-OH (or D ) CHz=N-OCHa CHz=N--N( CHa)z
7 63 t o 9 9.Y 6 96 to 9 22e 103102 117to12OiO2 116102
,
P
11.0t o 11.4O
i I
MezCO
12. i 0.2
J
I!
16.11 16.52 16.08 16.20 16.34 16,86 16.97
MeCR‘ (neat j Me2SO MeCS 16.5 ClCHzCHzCl CCla (neat) a All compounds were preDared in standard fashion by the reaction of formaldehyde with the appropriate Hth7-R compound. All .I values were measured on carefully calibrated Varian Associates Model A-60 spectrometers, radiofrequency 60 Mc. /see., sample temp. 36 1 2”. Unless otherwise specified, the P . E . of the values given is 0.05 C.P.S. or less. Unless otherwise noted, dilute (-5% or less) solutions were used. A rough “average” value, convenient for indicating the general sizes of the couplings. e Strongly solvent dependent. For a more detailed account, see B. L. Shapiro, S. J. Ebersole, and R . M. Kopchik, J . Mol. Spectry., 11, 326 (1963). Small concentration dependence. Small solvent dependence. See G. J. Karabatsos, B. L. Shapiro, F. M . \’am, J , S. Fleming, and J . S. Ratka, J . A m . Chem. Soc ,85,2784(1963). We thank Dr. P. L. de Benneville of the Rohm and Haas Co., Bristol, P a , for generous samples of this compound and for helpful discussions
side the range 0 f 3.5 C.P.S. only for the cases of substitution by elements of very low electronegativity, such as AI, Li, and 31g,3where values in the range (+) 6 to 8 C.P.S. are observed. In Table I, we report our observations of ~JHH,(gem)in a number of CH2=Ncompounds, in which very different values are obtained. Among other interesting features4 of the spectra, the magnitudes and (in some cases) solvent dependence~ of ~the J values are noteworthy. In these CH2= N--Y compounds, lJ1 increases markedly as t h e electronegativity of Y decreases. The trend observed here, taken toget.her with that reported for the oleJ fins,2h suggests that the sign of these CH2=N-Y values is negative, and various sign determination experiments are in progress. I t may be noted at this stage, however, t h a t the range of magnitudes of these sp2-typeJ(gem) values overlaps extensively with the range of values hitherto associated with sp3-type J(gem) values.5 Thus, regardless of the signs of the J values reported here, a theoretical picture which gives major importance to the H-C--H bond angles6 will clearly be inapplicable, since it is evident that in our sp2-typecases, substituert efects (to put the matter in the most general terms) are dominant, as has already been pointed out for sp3-type CH2 systems and suggested strongly for vinylic sp2cases as well.’ Regardless of the detailed nature of the dominant factors controlling the spin-spin coupling constants, the observed monotonic trend in iJl(gem) with p( 3 ) (a) 1) W hlotire and J A . Happe, J . Phya Chem , 66, 224 (1961); ( b ) C . S Johnson, Jr , M A Weiner, J S. Waugh, and 17 Seyferth, J . ,4m. Chem. Sor , 8 3 , 1306 (1961); ( c ) K . T. Hobgood, J r , and J H . Goldstein, Ypertvorhim A r l o , 18, 1280 (1962); (d) G Fraenkel. 1) C;. Adanis, and J Williams, T?lvahedvo?t L e t t e r s . No. 1 2 , 767 (1063) (4) To he discussed a t length elsewhere (a) See for example, b! Barfield and I ) . M . G r a n t , J A m . Chem. Soc., 86, 1x09 (1963), and reference.. cited therein (6) Cf M Karplus and I). H. Anderson, J . C h e m . P h y s . , 30, 6 (1959); H S. Gutowsky, M. Karplus, and I) M . Grant. i b i d . , 31, 1278 (1959); H. S . Gutowsky, V. I). Mochel, and B G. Somers, i b r d . , 36, 1153 (1962); M . Karplus. J 4 1 n (’hem S o c - . , 84, 2458 (1962). ( 7 ) H J Rernstein and N Sheppard. J . Chem. P h y s . , 37, 3012 (1962).
atom electronegativity (cf. vinyl-XZh) suggests that resonance form B, which is certainly unimportant in the azomethines, is also probably not very significant in the oxime and hydrazone derivatives. Resonance contributors of type A, however, could well be involved to some significant extent in all three types of compound. One possible and plausible implication of
+
-
CHz-X-Y A
CH 2-
+
N=Y B
this is that the more ‘(positive”the carbon atom of the CH2, the larger the ~ J l . However, other factors (such as the involvement of the n-system, and especially the availability of electron pairs on X of the CH2=X system) may well be important. These and other matters are currently being investigated. Acknowledgment.-It is a pleasure to acknowledge stimulating and helpful discussions with Drs. A . A . Bothner-By and P. C. Lauterbur. MELLON INSTITCTE B L SHAPIRO PITTSBURGH, PENNSYLVANIA S J . EBERSOLE KEDZIECHEMICAL LABORATORY G J KARABATSOS MICHIGAN STATECNIVERSITY F M VANE EASTLANSING, MICHIGAN SPACESCIENCE DIVISIOX S L MANATT JET PI?OPULSIO~X LABORATORY OF TECHNOLOGY CALIFORNIA INSTITUTE PASADENA, CALIFORNIA RECEIVED OCTOBER 18, 1963
Complexes of Organolithium Compounds with Vacant Orbital Acceptors. TI. Determination of ElectronDensity Changes by Proton Magnetic Resonance
Sir: We wish to report that changes in electron density of an aromatic organolithium compound, brought about by dative-bond formation with a Lewis acid, can be determined from changes occurring in its proton magnetic resonance ( p , m , r , ) spectra on complexing. Organolithium compounds form reversible dative con~plexes
