l l a y 5 , 1962
174’7
COMJIUNIC.\TIONS TO THE EDITOR
the reaction of tertiary phosphines with polyhalomethanes.4 (-1) Acknowledgment is made t o t h e Donors of the Petroleum Research Fund administered by the American Chemical Suciety ( G r a n t 286-A), and t o the National Cancer Institute of t h e Sational Institutes of Health (Grant CY-4769) for support of this research. ( 5 ) Alfred P. Sloan Fellow, 1981-1963.
DEPARTMEST O F CHEXISTRY FAUSTO RAhfIREZ6 STATEUSIVERSITYOF SEW TORK S.B. DESAI LONGISLASD CESTER S.MCKELVIE OYSTERBAY,S . Y. RECEIVED FEBRUARY 22, 1962
I%
+ 100
80% DIOXANE
70% Q
h b
cno/
‘i,O%
EFFECT OF SOLVENT ON T H E OPTICAL ROTATORY DISPERSION OF UNCHARGED MOLECULES CONTAINING T H E PEPTIDE GROUP’
Sir: It has been reported2a3that the optical rotatory dispersion of many native globular proteins does not resemble the optical rotatory dispersion of polypeptides with an a-helical structure, and that the change in going from a native to a denatured state is not the change expected for a helix-coil transition. In terms of the Moffitt-Yang equation4S5
/‘ 1
300
i t is found t h a t typical right-handed a-helical structures have ba ‘v -650° (with Xo assigned a value of 212 mp), whereas unfolded structures have bo ‘v 0. I n the many “non-helical” globular proteins, by contrast, bo is close to zero in both native and unfolded states. Only a. changes when the protein becomes denatured. ;Z possible explanation of these results is the existence of a specific structure, as yet unidentified, which all “non-helical” proteins have in common. An alternative suggestion2 is that no particular structure is needed to account for the observed results : that the di3erence between the optical rotation of native and denatured proteins may be partly (sometimes entirely) a quasi-solvent effect, reflecting the fact that the peptide groups of the native protein are largely in the interior of the globular structure, whereas in an unfolded conformation they are in a medium consisting largely of water. X necessary part of any proof for such a hypothesis is a demonstration that the optical rotatory properties of independent peptide groups depend strongly on the solvent. h?e have accordingly made measurements, in a variety of solvents, on several simple molecules which contain peptide groups. Only uncharged molecules were used, (1) This work was supported b y research grant G 17177 from the National Science Foundation, and by research grant .4-4376 from t h e National Institute of Arthritis and hIetabolic Diseases. U. S . Public Health Service. (2) C . Tanford, P. K . De a r d V. G. Taggart, J . A m . Chem. Soc., 82, 6028 (1960). ( 3 ) B. Jirgensons, Teti,ahedron, 13, 166 (1961). (4) W. Moffitt and J. T. Yang, PTOC.S a l . A c a d . Sci., U . S., 42, ,596 (1H,j6). I n this paper this equation is t o be regarded a? purely empirical. ( 5 ) I n equation 1 , [ C Y ] is t h e rotation a t any wave length A , .Me the molecular weight (in proteins it is t h e molecular weight per residue), ?I t h e refractive index of t h e solvent, and AO an absorption wave length, which bas been assigned a value of 212 mp in this paper. T h e parameters ao and bo are derived from experiment.
WATER I
1
400 500 Wave Length, rn”
608
Fig. 1.-Optical rotator)- dispersion of S-acetyl-Lglutamic acid, a t 25’, in dioxane-water mistures. Concentration of dioxane is per cent.by volume. .I Rudolph spectropolarimeter, with a mercury lamp as light source, was used.
so that changes in state of ionization (which are known to have important effects on the optical rotation of amino acids and small peptides) cannot occur. The results of one such study are shown in Fig. 1, which gives the measured rotation for N-acetyl-Lglutamic acid in mixtures of dioxane and water. It is seen that a striking solvent effect is indeed observed. \Then the data are plotted according to equation 1, with Xo = 212 m p , the values of a. and bo given in Table I are obtained. A change in solvent is seen to influence a. but not bo. The total TABLE I P A R A M E T E R S O F THE h,fOFF1TT-IwASG
EQUATIOS~ (Degrees
of Rotation) a0
bo
I n water -165 +67 I n l0Yc dioxane -140 +62 I n 20Yb dioxane - 113 +65 In 30y0 dioxane - 89 +68 In 40YGdioxane - 60 $67 In 50% dioxane - 28 +67 I n 60% dioxane G +64 I n 707, dioxane 35 +66 I n 8054 dioxane 64 +67 @-Lactoglobuline S a t i v e -169 -66 -Denatured -623 --il -!-Globulind S a t i ve -280 0 Denatured -600 -20 “ Using An = 212 m,u. The dispersion of refractive indes was taken into account in the calculations of this paper. Ac-L-G~u = S-acetyl-L-glutamic acid. Ref. 2. d C. Tanford, C. E. Buckley 111, P. K. De and E. P. Lively, J . B i d . Chem., 237, 1168 (1962). AC-L-GIU~
+ + +
1745
COXMUNICATIONS TO THE EDITOR
change in a0 in going from water to S07Gdioxane is approximately half as large as the difference in a0 between denatured and native “non-helical” proteins, data for two of which are shown in the table for comparison. T h a t these results are not due to the presence of a small number of ionized carboxyl groups in the purely aqueous solution, with suppression of the ionization as dioxane is added, was demonstrated by repeating some of the experiments in the presence of 0.1 M HC1. No significant change was observed. The general trend of the data is also independent of the choice of Xo, within narrow limits. m’ith Xo = 234 mp, for example, bo = 45’ and a. goes from -138 to + 5 3 O . Data similar to those shown here have been obtained with N-acetyl-L-leucine X-benzoyl-L-glutamic acid, and N-benzoyl-L-leucine amide, and with a wide variety of solvents For each substance, the effect of the solvent is to change ar and not bo. In each case the non-polar solvents lie a t one extreme and the more polar ones a t the other. The results will be reported in full a t a later date. The conclusion to be drawn from all the data is t h a t a change in environment of peptide groups of a protein, such as accompanies denaturation, is likely to make a substantial contribution to the over-all change in optical rotation, and that this contribution is likely to appear as a change in anwhen the data are analyzed by use of equation 1
I-01, s4
OH
1
AI, ’0
OCHj 1
TT
I1
1
Picropodophyllin
* y & 0 6 G o Ar
I11
0-
Ar
0
Iv
‘ Ar
0
v
Podophyllotoxin Test of this possibility required formation of the enolate of picropodophyllin. T o avoid complications arising from the presence of hydroxyl hydrogen, picropodophyllin (I) was converted by combination with dihydropyran t o O-tetrahydropyranylpicropodophyllin (11). One of the two crystalline diastereoisomers of 114 was treated a t room temperature with just over one molar equivalent of triphenylmethylsodium in ether.5 Then to neutralize enolate I11 in the resulting tan mixture, (6) T h e able technical assistance of Mrs P hl Hudson 15 ac cold acetic acid containing a trace of sulfuric acid knowledged was added in one portion. After preliminary DEPARTMEXT OF BIOCHEMISTRY fractionation of the protonated material, the DUKEUNIVERSITY CHARLESTAX FORD^ tetrahydropyranyl derivative (IV) of podophylloDURHAV, N C toxin was treated with hot hydrochloric acid in RECEIVED FEBRUARY 15, I962 alcohol to remove the protecting group. Crystallization and chromatography of the product furnished pure podophyllotoxin (V) in 23YGyield.6 SYNTHESIS OF PODOPHYLLOTOXIN’ Sir : Approximately 40% of the protonated product Podophyllotoxin (V), containing a strained consisted of the tetrahydropyranyl derivative of lactone system, is easily and essentially completely picropodophyllin (11) mixed with some picropodoisomerized with alkaline catalysts to the thermo- phyllin. It is expected that proton sources bulkier dynamically more stable picropodophyllin (I). than acetic acid will increase the ratio of podoWe now find that protonation of the enolate3 phyllotoxin to picropodophyllin. Since picropodophyllin has been synthesized,’ of a picropodophyllin derivative makes possible the regeneration of podophyllotoxin (17) from picro- the work described here completes a total synthesis (4) Some of the properties of this form are: m p. 203-204‘; lalz6D podophyllin (I). + l o 3 ( c = 1 in chloroform): ultraviolet absorption maximum a s a Protonation of the enolate of picropodophyllin 10-4 M solution in alcohol, 201 m e (log c 3 5 1 ) ; no infrared absorption (as in 111) could lead either to podophyllotoxin or for hydroxyl a t 3700-3126 cm.-’; A m i . Calcd. for CmHsnO?i, C, to picropodophyllin. Examination of models sug- 65.05, H , 6.07. Found: C , 65.31: H, 6.10. Exploratory work by gested that the front of the enolate is somewhat Dr. S. C. Chakravarti showed t h a t t h e reaction of podophyllotoxin with dihydropyran and hydrochloric acid catalyst gave t h e crude more accessible to a n approaching proton donor tetrahydropyranylpodophyllotoxin in low yield. Later, R . G . Rlcthan the back. Conceivably, therefore, frontside Innes obtained the tetrahydropyranyl derivatives of podophyllotoxin protonation, which would give podophyllotoxin, and of picropodophyllin, as mixtures. by using dihydropyran as s d v e n t could compete effectively with backside protona- and a trace of phosphorus oxychloride as catalyst. I n the present work, the tetrahydropyranyl derivatives of picropodophyllin were pretion, which would give picropodophyllin. pared with dihydropyran in chloroform solvent with fi-toluenesulfonic (1) This report. describing a portion of t h e Doctoral research of C. D. Gatsonis, represents paper No. XI11 in t h e series on “Compounds Related t o Podophyllotoxin”; the preceding paper is by W. J . Gensler, F. Johnson, and A . D. B. Sloan, J . A m . Chem. Soc., 82, 6071 (1960). ( 2 ) A comprehensive resierv is given by J. I,. Hartwell and A . W. Schrecker, Forfschr. Chem. O r g . .\-afwsfofe, 17, 83 (19.58) (3) T h e rate controlled protonation of enols has been studied carefully by Zimmerman and his associates Pertinent references may be found in a paper by H. E. Zimmerman and T . W. Cutshall, J . A m . Chem. S u c , 81, 1305 ( 1 9 5 9 ) . I n the present note, we have made no distinction between enol and enolate; the same stereochemical arguments apply to hoth.
acid as catalyst. ( 5 ) W. B. Renfreu-, Jr , and C. R. Hauser, “ O Y ~ .Syntheses,” Collective Volume 2 , 607 (1943); C. R . Hauser and B. E. Judson. J r . , O r g . Reacfioizs, 1, 286 (1912). (6) Some of t h e d a t a are: m.p. 160-161°; t h e synthetic podophyllotoxin showed m.p. 156-157‘ when mixed with authentic material ( m p. 156-1Ri0), [a]% - 1 3 2 O ( c = 1 in ch1.iroform); t h e infrared absorption curves of synthetic and authentic podophyllotoxin are the same, treatment of synthetic podophyllotoxin with piperidine causes isomerization t o picropodophyllin. ( 7 ) W .J. Gensler, C . .2.1 Samour. Shih Vi Wang and P J(,hnson, J . A m . ( ‘ h ~ m.?or , 82, li14 fl960).
