September, 1963
AMINOICYLADENISES
thyroacetic acids, were the most potent conipouiids tested. I n the test system employed, VI1 and XI11 were as potent as L-T,, one of the most active hypocholestereiiiic thyrominietic ageiit 3 reported in the literature. I t can be seen that replacement of the 3’-iodine atom i i i VI1 ~ i t h an isopropyl group has had no effect on hypocholesteremic activity (compare T’II with XIII). This findiiig is coiisisteiit with previous observations iioted with compounds containing an alanine side cliain.’O Although the number of compounds screened was quite small it appears that a structure-function relationship does exist. Maximum activity resides iii those compounds with a tn-o-carbon side chain arid a 3’-iodiiie atom or isopropyl group (VII, VIII, and XIII). Increasing or decreasing the length of the side chain or replacing the 3’-iodine or isopropyl group with Iiydiogrii decreases cholesterol-loirering activity. Formation of the methyl ether in most cases also somewhat lessens hypocholesteremic activity (compare T’II with T’III). ,i more comprehensive study of the thyroniimetic nctiITities of VI1 and XI11 has been reported rccently.*O
563
TABLE 111 PLASUA CHOLESTEROL VALUES .4 c tl v1t y a
Coinpound no.
I
0 030 001
ill causes \\ere deterniincd oii I (:orlcentrations and solvent eniployed were 5-7 X 1W6;I1in lnethanol. ethanol-JI sxrinioniuni acetate pH 7.5, 7: 3. HolvcLnt 13: methanol. Solvent C : iiietlianol c Solvent A : s 1 ~ 1 ~ t i o in x ~ methanol. s gl:,&il :iwtic*acid-water., 7'2: 3: 3. In all cases cinly one spot was observed. Spots irere loc,:iteti visually with an ult.rttviolt:t, l : t n i I l . ?J
2 LO
300
260
180 Wave l e n g t h , i
w .
320
-
September, 1963
did not yield any nitrogen with nitrous acid. h mixture of 1mole of adenine and 1 mole of phenylalanine yielded 2 moles of amino nitrogen, while 1 mole of Gly-Ad, L-Ala-Ad, and DL-Val-L$d each yielded 1.O mole of amino nitrogen. Slightly higher yields of nitrogen were obtained from L-Leu-Ad (1.2 moles), L-Ileu-Ad (1.2 moles), and L-Phe-Ad (1.1 moles). The excess nitrogen from the latter three compounds may be derived from partial hydrolysis of the aminoacyladenines in the acetic acidnitrous acid solution during deamination. It is evident that there are no free amino groups in carbobenzoxyaminoacyladenines and one free amino nitrogen in the aminoacyladenines. Of the various sites for substitution on the adenine molecule, these data support substitution on the 6-amino group. Deamination of adenine containing an amino acid substituent at any position but the 6-amino N would yield two moles of nitrogen per mole of compound. Treatment of carbobenzoxyaminoacyladenines with either N HC1 or iL' KaOH a t 100" for 1 hr. resulted in complete hydrolysis of the amide bond between the carbobenzoxyaminoacyl moiety and adenine. Milder conditions, 0.5 N KaOH for 20 min. a t loo", sufficed to hydrolyze aminoacyladenines to yield 1 mole of free adenine per mole of aminoacyladenine. Calculation of the extent of hydrolysis was based on the absorbance ratio 260/275 mp and is described in a subsequent section. The reaction was also followed by paper chromatography and, where possible, by measuring the increase in ninhydrin-reacting material during the heating pericd. The ultraviolet absorption spectra of aminoacyladeniiie solutions in water and in methanol changed on standing at room temperature for short periods of time. Since the compounds mere to be utilized for enzymatic htudies it was necessary to determine their stability iii aqueous solutions at various pH values. I n acidic media the absorption maximum of Gly-Ad is at 27*5 nip. -4fter 90 hr. a t pH 2 the peak a t 275 mp is replaced by a broad absorption band with a peak at 263 mp (Fig. 3). =It pH 12 (Fig. 4) the absorption peak of HOURS
no
240
565
~~~~I?~'o.~cI.L.~I)ENINEs
260
280
300
320
340
360
Wave length, mu.
Fig. 3.-Clianges in the ultraviolet absorption spectrum of 6.0 X 10-6 JI glycyladenine solution as a function of time a t pH 2 .
Gly-Ad at 280 mp is rapidly replaced by a peak at 269 mp, representing hydrolysis to adenine. Confirmation was obtained by chromatography of aliquots a t different time intervals; there was an increase in intensity of an
0.8
I
1
Ilr
220
240
260 280 300 Wave length, nip.
320
340
360
Fig. 4.-Changes in the ultraviolet absorption spectrum of a glycyladenine solution as a function of time a t 6.0 X 10-5 pH 12. 0.7 0.6
AO.5
.-
32 0.4
-
.3" 0.3
8
0.2 0.1
220
240
260 280 300 Wave length, mp.
320
340
360
Fig. 5.-Changes in the ultraviolet absorption spectrum of a 6.0 X 1 0 - 6 J I glycyladenine solution a3 a function of time a t pH 7 .
adenine spot, a decrease in intensity of the Gly-Ad spot, and an increase with time of a niuhydrin-reactive spot corresponding to glycine. At pH 7 (Fig. 5), major spectral changes in Gly-Ad occur in a region (310 mp) not associated with simple hydrolysis, the significance of n-hich has not been established. Stability studies mere also carried out at other pH values and with other aminoacyladenines. The results were not significantly different from those with Gly-Ad. Enzymatic Studies.-In order to establish whether aminoacyladenines may or may not exist in tissues, the susceptibility of these compounds to hydrolysis by various enzymes and tissue preparations was studied. Commercial preparations of pepsin, trypsin, a-chymotrypsin, papain, ficin, bromelin, pancreatin, acylase, and leucine aminopeptidase were incubated with aminoacyladenines. Hydrolysis was measured by the change in absorbance ratio 260/275 mp. As a standard for the calculation of hydrolysis, the absorbance of different proportions of pure aminoacyladenine and adenine in acid solution was measured a t 260 and 275 mp. The absorbance ratio 260/275 was determined for each proportion. This ratio is a constant for each pure compound; it varies linearly from 0.56 for aminoacyladenine to 1.71 for adenine and hence can be used to calculate the extent of hydrolysis of aminoacyladenines. Incubation mixtures of aminoacyladenines and enzyme were acidified after 1 hr. at 37" and the per cent of hy-
