\
r,
When s increases by As, the volume, V , increases in one direction by the same value and in three directions by 3As. At unit volume this variation is equal to the variation of density or specific gravity A8. Therefore AS = k43As = kl AS Substitution of this value in Equation 18 gives:
4
k2p + k3p2 = Tce-k18
02
+ klao[l - , - A s ] (19)
An identical relation holds for the variation of refraction An but with other values for kp and k3. Therefore klp 0.4
Figure 2.
f
kl8o[1
- e-An]
+
A8
Dividing Equations 19 and 20 by the constant factor T,e-k18 -t gives:
Temperature vs. intermolecular distance, S
with the corresponding values, s A s and s, and subtracting we have
T,[1 T,e-kIe
Tce-k18
(20)
5 in em: /o-'
-k
=
a
I
T2 - Ti = A T = T,[1 - e - k 1 ( 8
+ kip2
-
80)]
-
- e-k1(8 - I = + kieo (1 - ,-kiAs) 80)
(17) For a small AT the heat capacity is constant so that A T = k'q, proportional to the calories q added to the liquid. The mean distance, s, can be changed at constant temperature by dissolving a substance in the liquid, since the intermolecular forces between a solute and solvent molecule are different from those of two solvent molecules. If the number of solute molecules is relatively low, each of them will be surrounded by the same number of solvent molecules and therefore the variation 3 s is proportional to the weight per cent, p, of
k180
the solute: k2p. Furthermore, an intermolecular force acts continuously between a certain number of solute molecules which must also be considered. The effect on As of these forces must be proportional to the collisions between those molecules which is proportional to the square of all the solute molecules present , N , times their molecular weight, M , or to per cent, p.
k,(M11')2 = k3p2 With the greater M value, the two molecules remain together longer after collision. The total contribution of the py0 solute in the solution which determines the variation As is therefore the k3p2. sum k2p Substitution in Equation 17 gives: k180(1 - e - k l b ) k2p + k3p2 = T,e-k"
+
(18)
1
- e - A S = a o p + bop2 1 - e - A n = np + b p 2
(la)
(1)
These equations agree exactly with experimental data a t constant temperatures and for the indicated range of concentration. When Equation 16 is applied for associated liquids, a correction must be introduced if Equation 16 is used from 0' K., up to T , . For a given range of temperature where the degree of association varies linearly with temperature, Equation 16 holds exactly for this range. For the derivation given above, constant temperature is presumed and therefore Equations 1 and la are valid also for associated liquids-e.g., water, ethyl alcohol, and methyl alcohol. RECEIVEDfor review January 7, 1963. Accepted December 13, 1963.
A Spectrophotometric Test for Xanthopterin WALTER S. McNUTT Department of Pharmacology, Tufts University School of Medicine, Boston, Mass. 02 7 7 7
b The structural requirement of a simple pteridine for a positive test in the Folin-Ciocalteau color reaction appears to be an appropriately substituted 6-hydroxypteridine. The test serves to distinguish pteridines related to xanthopterin from those related to isoxanthopterin. The test may thus be applied in studies on the structure of naturally occurring pteridines.
EXPERIMENTAL
X
gives a characteristic blue color with the Folin-Ciocalteau reagent, whereas isoxanthopterin is negative in this test. The test may be of use in studies on the structure of naturally occurring pteridines. ANTHOPTERIN
912
A study has been made of the substituents which contribute to the ease of oxidation of the pteridine by this reagent. Among the simple pteridines, the structural requirement for a positive test (that is, ease of oxidation) appears to be a properly substituted 6-hydroxypteridine. 6-Aminopteridines have not been examined.
ANALYTICAL CHEMISTRY
Materials. Xanthine-8-carboxylic acid was synthesized (8). Guanine-8carboxylic acid was prepared by an analogous synthesis. The other purine derivatives and the following compounds were commercial producb:
xanthopterin, folic acid, and p-amino benzoyl glutamic acid. The 2-, the 6-, and the 7-monohydroxypteridines and the 2,6-, the 2,7-, the 4,6-, and the 4,7-dihydroxypteridines were obtained from A. Albert. 6-Methyl-2,4,7trihydroxypteridine and the N-methylpteridines were obtained from W. Pfleiderer. 2 - Amino - 4 - hydroxypteridine-&aldehyde was obtained from G. Brown. The other compounds were synthesized and shown to be authentic on the basis of their ultraviolet absorption spectra. Care was taken in the purification of the pteridines, as 4 , s diaminopyrimidines give a strong color development with the Folin-Ciocalteau reagent. REAQENTI. 110 ml. of aqueous
solution containing 1O grams of NaZCOI. REAGENT11. AqL.eous solution (100 ml.) containing 10 grams of Na2C03 and 1.25 grams of sodium tartrate plus 10 ml. of 0.50/6 CuSOa solution. [See Lowry et al. ( I ) ] Procedure. One milliliter of reagent was added t o 5 ml. of solution
Table 1. Comparison of Purine Derivatives and Monohydroxypteridines in the Folin-Cicicalteau Test
Purine or pteridine derivative
REagent I, % trans- Reagent 11, % mit- transmittance tar ce for for 1 1 2 umde pmole Nmoles per per per tube tube tube
0
Uric acid Xanthine Guanine Isoguanine Guanine-8carboxylic acid Xanthine-8carboxylic acid Guanosine Caffeine Adenine Hypoxanthine 8-Azaxanthine S-Azaguanine 7-Hydroxypteridine 6-Hydroxypteridine 2-Hydroxypteridine
22
30
45 95
56 93 99
100
39
>loo >loo >loo >loo 100 99
98
88
>loo >loo >loo
>loo
98
97
Table II. Ease of Oxidation of Pteridines Disubstituted with Hydroxyl or Amino Groups
Reitgent
I,
Pteridine derivative 0 Lumaxine (2,4dihydroxy pteridine) 2,&Dihydroxypteridine 4,6-Dihydroxypteridine 6,7-Di hydroxypteridine 2,7-Di hydroxypteridine 4,7-Dihydroxypteridine 2-Amino-4-hydroxypteridine 2-Amino-4-hydroxypteridine-6-carboxylic acid
1%
(water or dilute Na2C03)containing 1 or 2 pmoles of compound. T h e solutions were mixed well and allowed to stand at room temperature for 10 minutes. Folin-Ciocalteau reagent, 1N with respect to acid (0.5 ml.), was added and mixed immediately. The per cent transmittance at 500 mp in a cylindrical tube 12 mm. in diameter was recorded in a Bausch and Lomb colorimeter (Model 340) after standing for 30 to 40 minutes.
Comparison of monohydroxypteridines with certain purine derivatives (Table I) shows that 6-hydroxypteridine itself is negative in this test. Substitution of 6-hydroxypteridine with a hydroxyl group at position 2 (Table 11) satisfies the structural requirement for maximal color development in comparison with purine derivatives, That the requirement for the ease of oxidation is a substituted 6-hydroxypteridine rather than a substituted 2-hydroxypteridine, is shown from the fact that 4,6-dihydroxypteridine undergoes some oxidation by the reagent, whereas 2hydroxypteridines other than 2,6dihydroxypteridine are not attacked (Table 11). Pteroic acid, folic acid, and “2hydroxyfolic acid” give rise to some development of color (Table 111). Part of the color development may be due to the p-aminobenzoic acid portion of the molecule (Table 111). As this portion of the molecule accounts for only part of the color development, partial replacement of the side chain at the 6-position by a hydroxyl group may also occur. 2-Amino-Chydroxy-
14
13
74
72
>loo
99
9!)
97
99
98
100
96 100
Reagent I, % trans- Reagent 11, yo mit- transmittance for tance for 1 1 2 pmole pmole pmoles Per per per tube tube tube
0 100 Xanthopterin (2-amino-4,6d1hydrox.ypteridine) 13 2,4,6-Trihydroxypteridine 14 2-Amino-6,7dihydroxypteridine 14 4,6,7-Trihydroxypteridine 12 7-Methyl-2,4,6trihydroxy17 pteridine Xanthopterin-7carboxylic acid 60 Isoxanthop terin (2-amino-4,7dihydroxypteridine) 97 2,4,7-Trihydroxypteridine 97 6-Methylisoxanthopterin Isoxant hopterin6-carboxvlic acid 6-Methyl-2,4,7trihydroxypteridine > 100
100
100
19
5
12
3
10 25 17 58 97
92
96
93 91 94 94
100
Table V. Ease of Oxidation of Pteridines Related to Tetraoxypteridine Table 111. Oxidation of Compounds Related to Folic Acid by the FolinCiocalteau Reagent
Compound 94
Pteridine derivative
RESULTS AND DISCUSSION
trarls- Reagent 11, % transmittance tan’:e for for 1 1 2 pmclle pmole @mole per per per tube tube tube 100 100 100 93
Table IV. Ease of Oxidation of Pteridines Trisubstituted with Hydroxyl or Amino Groups
0 Pteroic acid Folic acid Rhizopterin 10-Formylfolic acid "2-hydroxy folic acid” lO-Nitroao-“2hydroxy folic acid” 2-Amino-4-hydroxypteridine-6-aldehyde p-Aminobenaoylglutamic acid
Reagent I, % trans- Reagent 11, % mib transmittance tance for for 1 1 2 pmole pmole @moles per per per tube tube tube 100 100 100 36 32 71 62 30
>loo >loo >loo >loo 69
64
37
>loo >loo 99
96
80
81
65
Pteridine derivative 0 Leucopterin (2amino-4,6,7trihydroxypteridine) 2,4,6,7-Tetrahydroxypteridine 1-Methyl-tetrahydroxypteridine 3-Methyl-tetra: hydroxypteridine %Methyl-tetrahydroxypteridine 1,3,&Trimethyltetrahydroxypteridine
Reagent I, % trans- Reagent 11, % mit- transmittance tance for for 1 1 2 pmole pmole pmoles per per per tube tube tube 100
100
100
22
18
2
17
12
2
25
19
11
12
21
21
6
4
VOL. 36, NO. 4, APRIL 1964
0
913
pteridine-6-aldehyde (Table 111) and 2amino - 4 - hydroxypteridine - 6carboxylic acid (Table 11) are negative in this test, and methyl substitution at the 6-position of this pteridine would not be expected to lead to a positive test. Protection of the amino group a t the 10-position with a formyl or nitroso group renders the compounds resistant to oxidation by this reagent (Table 111). A comparison of pteridines related to xanthopterin and isoxanthopterin is shown in Table IV. The positive test of 2,4,6-trioxypteridine is but little affected by substitution of a methyl group at position 7, but substitution of a carboxyl group a t the 7-position of xanthopterin is associated with a reduction in the intensity of color development (Table IV). The production of color by 4,6,7-trihydroxypteridine shows that the structural
requirement for ease of oxidation is a properly substituted 6-hydroxypteridine rather than a substituted 2-hydroxypteridine. I n contrast to the effect of N-methylation on the response of xanthine in this test (caffeine is negative; Table I), N-methylation of tetrahydroxypteridine does not greatly affect the ease of oxidation of the pteridine (Table V). The color produced by 1,3,8-trimethyltetrahydroxypteridine actually exceeds that of tetrahydroxypteridine itself. KO attempt has been made to characterize the product of oxidation of the pteridine by this reagent. Oxidation of uric acid and leucopterin by chlorine has been shown to yield glycols in which the hydroxyl groups are introduced a t the junctions of the two rings (3, 4).
ACKNOWLEDGMENT
I am indebted to Adrien Albert for his helpfulness and for gifts of monoand dihydroxypteridines. I thank Wolfgang Pfleiderer for the N-methylpteridines, Gene M. Brown for the 2 - amino - 4 - hydroxypteridine - 6 aldehyde, and Roberta McDuffie for technical assistance. LITERATURE CITED
(1) Lowry, O., Rosebrough, IC. J., Farr, A. L., Randall, R. J., J . Bwl. Chem. 193, 265 (1951). (2) McNutt, W. S., Zbid., 238, 1120 (1963). (31 Wieland, H., Koteschmar, A., Annulen 530, 152 (1937). (4) Wieland, H., Meteger, H., Schopf, C., Bulow, M., Zbid., 507,226 (1933). RECEIVED for review January 6, 1964. Accepted January 31, 1964. Work supported in part by Grant No. AM43675 from the U. S. Public Health Service.
A Study of Base-Catalyzed and Salt-Catalyzed Acetylation of Hydroxyl Groups GEORGE H. SCHENK, PATRICIA WINES, and CAROLYN MOJZIS Department o f Chemistry, Wayne State University, Detroit 2, Mich.
b Triethylenediamine [ 1,4-diazabicycIo(2,2,2)octane] is superior to pyridine for base-catalyzed acetylation of organic hydroxyl groups. Acetylation at reflux temperatures of most primary and secondary alcohols, phenols, and 1 -dodecanthi01 i s quantitative in 15 to 20 minutes. Most amines react at room temperature within 15 minutes. Triethylenediamine-catalyzed acetylation on a semimicro scale is suitable for the determination at room temperature of primary alcohols, some polymers, and some mixtures. Various mechanisms of base-catalyzed acetylation are also discussed. Although no physical evidence exists for an intermediate in pyridine-catalyzed acetylation, infrared, refractive index, and nuclear magnetic resonance studies established the existence of a triethylenediamineacetic anhydride complex in various solvents. A study of salt-catalyzed acetylation indicated that 0.544 tetraethylammonium bromide gives quantitative acetylotion at reflux temperatures of cyclohexanol in five minutes and tertiary alcohols in 45 to 65 minutes.
T
OF REAGENTS for the determination of organic hydroxyl groups has widened considerably since Mehlenbacher’s review (14) of this field. As recently compiled by Siggia HE CHOICE
914
*
ANALYTICAL CHEMISTRY
( l y ) , this choice includes, in addition to base-catalyzed acetylation and phthalation, acid-catalyzed acetylation and the base-catalyzed reactions of 3,5-dinitrobenzoyl chloride and pyromellitic dianhydride. Interestingly enough, pyridine still remains the solvent and/ or base catalyst for all of these methods. It is not difficult to appreciate why since pyridine is tit the same time a good enough nucleophile to catalyze the ionixation of acetic anhydride, and a poor enough base to avoid interference a t the end point with the basic titrants used to assess the per cent reaction. If any better nucleophile is to replace pyridine, it obviously must not be used as a solvent, but must be used a t a low enough concentration so that it will be conveniently titrated in a titrimetric finish such as that below: Ac20 PhNHz -C PhNHAc HOAc (1)
+
PhNH2
+
+ HC104 + PhNHaC104
(2) The excess acetic anhydride is removed by reaction with excess aniline and the excess of the aniline is titrated with standard perchloric acid in a nonaqueous medium. Few strong bases possess the steric requirements necessary for effective catalysis a t the low concentrations demanded by reactions l and 2. However, triethylenediamine [l14-diazabicyclo-
(2,2,2)octane], a bicyclic bridgehead diamine, is known to be a far superior catalyst than pyridine or triethylamine for the isocyanate-alcohol reaction (3,4 ) . Hanna and Siggia (1.2) have made analytical use of this catalysis. The mechanism (2) of this reaction as shown below closely resembles the chief mechanism of base-catalyzed acetylation given elsewhere. R-N=C=O
+ :NR3 G R-N=C-O-
I
(3)
@NR3 R-N=C-O-
I
+ :OR
+
H
@NR3 R-NH-CO2R
+ :NRI
(4)
The fact that triethylenediamine was found to have seven times the catalytic activity of triethylamine and almost 100 times the catalytic aetivity of pyridine in the above process (4), suggested that it would make an excellent catalyst for the acetylation of alcohols. This led also to a broad investigation of catalysis by sodium acetate and other salts where other mechanisms seemed to predominate. Three methods were studied in detail and are presented below: a macro method employing 2.5M acetic anhydride-0.19M triethylenediamine, a semimicro method em-
