2122
R. BJORKLUND AND SIDNEY KATZ
Vol. 78
TABLE I1 METAPERIODATE OXIDATION DATA __ Moles 1 0 4 - consumed per mole of Within 3 minutes
Uridine S ylopyranosylthymine X ylofuranosylthymine "Ribofuranosylthymine" .I Ribofuranosylthymine H Ribofuranosylthymine C Glucopyranosylthymine Thymidine
0.92 112 90
80
1 hr.
4 hr.
1 .OO 0.15 0.08
1.00 .. 0.14
..
1 .03
1 .03
. .
8 hr.
24 hr.
0.73
1.58 0.58 1.03 1.08
compound-
1 . (15
1.01 2.02
. 00
-
(36) b:. I.. Jackson and C. S.Hudson, T H I SJ O U R K A L , 5 9 , 9 9 4 (1937). ( 3 7 ) B. Lythgoe and A K . Todd, J . Chrm. SOL.,692 (1944). (38) E . I.. Jackson and C S. Hudson, THISJ O W R K A L , 61, 1530 (1939).
~
--
-
--- ~- .
7%hr.
2.10 0.79
2.20 1 01
1 ,05
. .
0.04
1.05
0.96 .29
Metaperiodate Oxidation Studies.-Concentrations of nucleosides ranging between 0.001 to 0.002 mM/ml. were treated with excess metaperiodate and aliquots titrated iodometrically according to the usual procedures.36~37 The acidity produced was determined according t o Jackson and Hudson.38 The results are listed in Table 11. The extent of the oxidation within three minutes is noteworthy, and would seem to correlate with the presence of cis-hydroxyls.
-
4 8 hr.
$10 h r .
. .
..
2.08
Acknowledgments.--Tlle authors are indebted to D ~J.. 0 . L~~~~~ for samples and for enzymatic Visser for assay Of Our compounds, to Dr. D. the sample of "ribosythylnine" h, and to Schwarz Laboratories, Inc., for a supply of crude guanosine. . ~ ~ ~ford The authors wish to thank D ~A4aron helpful discussions and Iris Wempen and John Vitals for valuable assistance.
w.
XEU.
YORK
21,
N E W YORK
[CONTRIBUTION FROM THE I)Oh-hEK LABORArORY O F MEDICAL P H Y S I C S 4 N D rHE v I R U 5 LABORATORY, UNIVERSITY 01: CALIFORNIA, BERKELEY]
The Molecular Weights and Dimensions of Some Human Serum Lipoproteins BY
I is the average value of KcIR90,". Average values were used in the calculations, because inspection of the scattering data (including that for 45 and 135') showed that Kc/Re, had no statistically significant tendency to increase with increasing c when the same combinations of neutral density filters were used. The values of P(90) were taken from a plot of P(90) vs. 2 computed from the particle scattering factor for spherical particles. In this range of 2, P(90) is very nearly independent of the model used and therefore the molecular weights are independent of the assumption of spherical shape. With molecular weights, flotation coefficients and dissymmetries available it was of obvious interest to examine the internal consistency of these quantities by calculating flotation coefficients from the measured molecular weights, dissymmetries and the "densities" of the hydrodynamic units. The last mentioned quantity was taken to be do, the density of zero sedimentation. For the purposes of these calculations we assumed no preferential adsorption of either buffer or water.22 The densities and the molecular weights were used to calculate the molecular volumes of each species. The diameters, D,of spheres having these volumes are shown in column 9 of Table 11. Flotation coef(22) S. Katz and H. K.Schachman, Biockim. Biopkys. Acta, 18, 28 (1955).
CrU' \
32
Y O
L
Y
PH l
c
I
3'
30
n 9.6
0 49 8 I
0
I
30 I
2
I
I
I
I
3
4
c(gm/ml)x'lO , Fig. 3-Plots of Kc/Rw,, X IO7 vs. concentration for the Sot 8.1 fraction a t PH 3.0, 4.9 and 9.6. The horizontal line is the average value of Kc/Rw,, X IO7 for this fraction a t PH 6.7.
ficients for spheres with these densities and volumes are shown in column 10. Using the same molecular volumes we calculated the dimensions of prolate ellipsoids of revolution having the measured dissymmetries using a relation derived by D e b ~ e . ~ ~ In Table 11, b is the length of the semi-minor axis and a is the length of the semi-major axis determined this way. With these dimensions, flotation coefficients can be calculated using the relation
where f/fo is the frictional ratio for prolate ellipsoids of revolution calculated using Perrin's formula and fo is the frictional factor for spheres. The density of the solution, d, is 1.0630 g./ml. The results are shown in columns 10-14 of Table 11. Evidently, granting the assumption of no preferential adsorption, only the ellipsoidal shape is consistent with the measured flotation rates, densities, dissymmetries and molecular weights. Since the dissymmetries are much too low for rod-like or coil-like molecules of these molecular weights these models are also ruled out. To illustrate that the molecular weights are virtually independent of the assumption of spherical shape used to calculate P(90), i t can be stated that (23) Cited in "Advances in Protein Chemistry," Vol. 6, Academic Press, New York, N. Y.,1951, p. 324.
2126
R. W. YOUNG,K. H. WOOD,R. J. JOYCE AND G. W. ANDERSON
this factor when calculated for the ellipsoidal models given in Table I1 differs by less than 0.02% from the P(90)for spheres having the same dissymmetry. It is difficult to compare our results with some of the earlier studies of human serum lipoproteins. On the basis of ultracentrifugal and diffusion measurements Pedersen6 obtained the value 2.6 X lo6 for what he assumed to be the “hydrated” molecular weight of “X-protein.” His estimate assumes that the partial specific volume of the sedimenting unitz2 (which may consist of protein plus associated water and salt) is independent of the salt concentration. This assumption is particularly critical in calculating a molecular weight since the partial specific volume was determined to be 0.97 and an error of only 0.01 in this figure would give an error of about 30y0 in the molecular weight calculated from the usual Svedberg equation. The light scattering method is, of course, free of this difficulty. In addition more recent ultracentrifugal s t ~ d i e s ~ have ,~ shown that Pedersen’s “X-protein” probably consisted of molecules with flotation constants ranging a t least from Si 4 to Sf 10. IYithout more information one can only say that there is no necessary disagreement between our results and Pedersen’s. Oncley, Scatchard and Brownz4have obtained an (24) J L Oncley, G Scatchard and A Brown, J P h y s Colloid Chem , 51, 184 (1947)
[CONTRIBUTION F R O X THE
Vol. 78
anhydrous molecular weight of 1.3 X lo6 for PIlipoprotein, a lipoprotein whose ultracentrifugal characteristics are similar to our 5’; ti fraction However, the degree of ultracentrifugal homogeneity of this preparation has not yet been reported, and as the preparation of P1-lipoprotein involved ethanol fractionation and dialysis (the latter having been shown to effect irreversible changes in serum lipoproteins)26a comparison of our results and those of Oncley, Scatchard and Brown appears unwarranted. Lindgren’ has calculated minimum molecular weights for some lipoprotein fractions using ultracentrifugal data and assuming the molecules to he spherical. This method of calculation applied to our $’ 8.1 and S,“ 6.4 fractions gave molecular weights which were 10% smaller than those calculated from the light scattering data. Acknowledgments.-It is a pleasure to thank Professor John W. Gofman for his interest in and encouragement of this work. We also wish to thank Professor Howard K. Schachman and Dr. Verne Schumaker for helpful criticisni o f the manuscript.
-
(25) B R Ray, B 0 Davisson and H L Crespi, :bid
68, 841
( 1 954)
BERKELEY,CAL
MEDICINAL CHEMICAL RESEARCH SECTIOS, RESEARCH DIVISIOS,AMERICAN
CVANAMIn
COMPANY ]
The Use of Phosphorous Acid Chlorides in Peptide Synthesis1 BY RICHARD TIT‘. YOUNG,KATHRYN H. WOOD,R.
JANICE JOYCE A X D
GEORGE\V. ANDERSON
RECEIVED SEPTEMBER 2, 1955 The preparation of peptide derivatives using ethyl dichlorophosphite and ethylene chlorophosphite under varying conditions is reported. The best conditions involve the addition of the chlorophosphite to a suspension or solution of the N-acylamino acid or peptide and amino acid or peptide ester in diethyl phosphite containing triethylamine The novel use of a trialkyl phosphite as an acid-acceptor in these peptide-forming reactions is described. The methods have been investigated for extent of racemization.
In this paper we wish to report the use of some In previous papers of this series the use of diethyl chlorophosphites and a study of various conc h l o r ~ p h o s p h i t e ,o-phenylene ~~~ c h l o r ~ p h o s p h i t e ~ other ,~ and tetraethyl pyrophosphite* for the synthesis of ditions under which the reagents may be employed peptides was reported. Since these publications ap- for peptide synthesis. Continuing the investigation of simple reactive peared, several reports on the use of these reagents for the synthesis of peptides of asparagine and glu- halophosphites, ethyl dichlorophosphite (I) was t a n ~ i n earginine6 ,~ and of oxytocin1 have been pub- prepared. This reagent may be prepared in large lished. C,H,OP< c1 I (1) Fourth paper in a series on phosphorus derivatives.
Portions
of this paper were presented a t t h e September, 1951,Meeting of the
American Chemical Society. In order t o maintain consistent nomenclature with previous papers in this series, t h e currently accepted names for phosphorus compounds (Chem. Eng. News, 30, 4515 (1952)) are not being employed. (2) G. W. Anderson, J. Blodinger, R . W. Young and A. D. Welcher, THISJ O U R N A L , 74, 5304 (1952). (3) G.W, Anderson and R. W. Young, ibid., 74, 5307 (1952). (4) G. W. Anderson, J. Blodinger and A . D. Welcher, i b i d . , 74,
5309 (1952). (5) H.K.Miller and H. Waelsch. Arch. Biochcm. B i o p h y s . , 353 176 (1952); S. J. Leach and H. Lindley, A u s . J. Chem., 7, 173 (1954); A. Miller, A. Niedle and H. Waelsch, Arch. Biochcm. Biophys., 6 6 , 1 1 (1955). (6) G. W. Anderson, Tam JOURNAL, 75, 6081 (1953). (7) V. duvigneaud, C. Ressler, J. M . Swan. C W Rnherts and P. G . Katsoyannis, i b i d . , 76,3115 (19.54).
c1
quantities from ethanol and phosphorus trichloride without using a tertiary base. Both halogens in I are reactive, the compound being both more stable (thermally) and reactive than diethyl chlorophosphite. The reagent may be employed exactly as are the monochlorophosphites; Le., I may react first with the acylated amino acid to form the “mixed anhydride” I1 which then may be used to acylate the amino ester (equation l), or the amino ester may first be converted to the “phosphite amide” I11 which then may be treated with the (8) N hlenschutkin A n n , 139. 343 (18flG).
