July 5 , 1968
2’(OR
3’) -0-(DL-PHENYLALANYL)-RIBONUCLEOSIDES
six peaks had been eluted, the total nucleotidic material was eluted with 1 M triethylammonium bicarbonate. The 1 M eluate (about 30% of material applied to the column) was evaporated t o dryness and the process of dissolution in water and evaporation repeated t o remove most of the salt. The total mixture was then chromatographed on a 9’’ wide strip Whatman No. 40 paper using solvent C. Each of the peaks obtained u p t o fraction 96 was also recovered by evaporation of the appropriate fractions. Characterization of the products recovered from the different peaks and the chromatogram was as follows. Cj’,-Pyridinium uridine-3’ phosphate (XX)(4.4% was in fractions 28-31, peak 2), was eluted at a 0.05 M triethylammonium bicarbonate concentration; ultraviolet absorption: Xmsx 260 mp, shoulder a t 267 mp; Ami, 232 mp in water; Amax 260 mlr shoulder a t 267 mp; and Amin 243 mp at pH 11.5. Paper chromatographic and electrophoretic mobilities are in Tables I and 11. Uridine-3’,5’ cyclic phosphate (7.5%) constituted peak 3, being eluted in fractions 32-34. The paper chromatographic and electrophoretic mobilities are in Tables I and 11. In its behavior t o acid and alkali and in its resistance to pancreatic ribonuclease, the product was identical with the sample synthesized earlier.26 Uridine-3’ phosphate (9.7%) was present in peak 4 (fractions 42-52) being eluted at 0.079 M triethylammonium bicarbonate conceritration, Uridine cyclic dinucleotide (5.20/) was present in peak 5 (fractions 58-64) being eluted at 0.082 M triethylammonium bicarbonate concentration. The paper chromatographic and electrophoretic mobilities (Tables I and 11) were consistent with its structure. Treatment with bacterial phosphomonoesterase under t h e standard conditions did not alter its electrophoretic mobility at pH 7.1. Incubation with pancreatic ribonuclease caused only partial degradation to uridine-3’ phosphate under the conditions which caused complete degradation of the dinucleotide uridylyl(3’+5‘)-uridine-3’ phosphate t o the mononucleotide. Treatment with 0.5 N sodium hydroxide a t 25” caused only abqut 20% ,hydrolysis in 6 hr. and about 6 5 7 0 % in 54 hr., while uridylyl-(3 -+ 5‘)-uridine was completely hydrolyzed in 6 hr. under the above conditions. The main product from the cyclic dinucleotide on alkaline hydrolysis was uridine-2’(3’) phosphate, there being detected a small amount of an intermediate, presumably linear uridine dinucleotide. Uridylyl-(3’+5’)-uridine-3’ phosphate (XVIII, n = 0) was present in peak 6, being eluted in fractions 84-98 (0.091 Af triethylammonium bicarbonate concentration). The position of
1997
elution, paper chromatographic and paper electrophoretic properties (Tables I and 11) were all consistent with its structure. Treatment with bacterial phosphomonoesterase caused complete conversion t o uridylyl-(3’-+5’)-uridine. The latter was identical in paper electrophoretic mobility with a synthetic sample and gave, after incubation with pancreatic ribonuclease, uridine-3’ phosphate and uridine in equal amounts. The dinucleotide itself on incubation was completely degraded t o uridine-3’ phosphate ~. with pancreatic ribonuclease. Uridine Cvclic Trinucleotide (XIX, n = l).-The material eluted by 1 A? triethylammonium‘bicarbonate after elution of the preceding compound, uridylyl-(3’+5’)-uridine-3’ phosphate, was separated by paper chromatography in solvent C on a sheet of paper (Whatman No. 44). The cyclic trinucleotide constituted a major band43 (Rr 0.25, solvent C ) of the 1 M fraction (about 3.5% of total polymeric mixture). Its paper chromatographic and electrophoretic properties are given in Tables I and 11. The electrophoretic mobility was almost identical with the previously synthesized thymidine cyclic dinucleotide. It was resistant t o the action of the alkaline phosphomonoesterase. Degradation with pancreatic ribonuclease gave a product which was identified as uridine-3’ phosphate. (Sufficient material was not available for a comparative rate study with known substrates.) Uridylyl-(3’+5 ’)-uridylyl-(3’+5 ’)-uridine-3’ phosphate (XVIII, n = 1) was present (about 2% of total nucleotidic material) in a band traveling slower ( R f0.15, solvent C ) than the cyclic trinucleotide. Its Chromatographic properties are in Tables I and 11. On incubation with the alkaline phosphomonoesterase it was completely converted t o a product (Rr 0.25 in solvent C ) which was identified as uridylyl-(3’+5’)-uridylyl-(3’-.5’)-uridine. Degradation of the latter with pancreatic ribonuclease gave uridine-3’ phosphate and uridine in a ratio close to 2. The trinucleotide itself on incubation with pancreatic ribonuclease gave only uridine-3’ phosphate. Higher Oligonucleotides.-Some 3% of the total polymeric mixture did not leave the origin on paper chromatography in solvent C as used above for separation of the di- and trinucleotides. This material evidently consisted of several components as shown by paper electrophoresis and included the oligonucleotides higher than those characterized above. (43) This product was further purified by paper electrophoresis at pH 7.1. I n this way two minor impurities were removed.
ENZYME RESEARCH, UNIVERSITY OF WISCONSIN, MADISON, WIS., A N D DIVISIONO F BRITISH COLUMBIA RESEARCHCOUNCIL, VANCOUVER, CAN.]
j C U N ~ l ’ K I t l U 1 . I U S FROM THE INSTITUTE FOR
THE
CHEMISTRY
Studies on Polynucleotides. XX.’ Amino Acid Acceptor Ribonucleic Acids (1). Synthesis and Properties of 2’(or 3’)-0-(DL-Phenylalanyl)-adenosine, 2’(or 3 ’)-0-(DL-Phenylalany1)-uridineand Related Compounds2
The
BY D. H. RAMMLER~ AND H. G. KHORANA RECEIVEDDECEMBER 11, 1962 ‘I’ht. reactiuti of carbobenzyloxy-DL-phenylalanine with dicyclohexylcarbodiimide gave a n excellent yield of the corresponding symmetrical anhydride 111, which was isolated in a crystalline form and characterized. The pyridine-catalyzed reaction of I11 with 5‘-0-tri-p-methoxytrityluridine followed by a n acidic treatment gave Inono-O-(carbobenzyloxy-DL-phenylalanyl)-uridine,which was, presumably, a mixture of the 2’- and 3’-isomers. Analogous Palladium-catalyzed hydrogenolysis of the latter afforded 2’(or 3’)-O-(~~-phenylalanyl)-uridine. reaction of 111 with b’-O-trityladenosine gave both 3’-O-(carbobenzyloxy-~~-phenylalanyl)-5’-O-trityladenosine and the corresponding 2’-isomer which were separated by chromatography on a silicic acid column. The isomers were found t o undergo interconversion under mildly acidic or basic catalysis. It proved possible, however, to determine the orientation of the protected aminoacyl residue in the products by phosphorylation and identification of the resulting adenosine-2’ or -3‘ phosphate. Removal of the protecting groups from the above derivatives gave DL-phenylalanyladenosine which was, presumably, a mixture of the 2’- and 3’-isomers. The rate of hydrolysis of the aminoacyl nucleoside was determined in pH 7 phosphate buffer. The half-life at 25’ was 48 rnin., while that a t 34” was 22 min. The possible causes for the great lability of the aniinoacyl linkage in the adenosine ester are discussed. Furthermore, the present work suggests the rapid migration of the amino acyl groups between the 2’- and 3‘-hydroxyl groups of terminal adenosine in amino acid acceptor ribonucleic acids.
’lhe first steps in the enzymatic synthesis of a polypeptide chain are the activation of a-amino acids, by ( 1 ) Studies on Polynucleotides. X I X : u. H . Rammler, y . I,apidot and H G . Khorana, J . A m . C h e m SOC.,86, 1989 (1963). (2) This work has been supported by grants from the National Cancer 1 nstitutes of the National Institutes of Health, the National Science Founds. LIuw Washington. 1). C.,and the Life Insurance Medical Research Fund, S e w York, N. Y. ( 3 ) U . S. Public Health Service postdoctoral fellow 19-59-1961. Present ‘iddress: 1,aboratory of Molecular Biology, National Institute of Neurolwical Ijiseases and Blindness, National Institutes of Health, Bethesda, Md.
reaction with adenosine-5‘ triphosphate, to form the mixed anhydrides of the type I and the subsequent transfer of the aminoacyl groups to the terminal adenosine residues (partial structure 11) of relatively molecular weight designated Variously as soluble, transfer, or amino acid acceptor (4) For the system of short hand representation of polynucleotides see H. G. Khorana “Some Recent Developments in the Chemistry of Phosphate Esters of Biological Interest,” John Wiley and Sons, Inc., New York, N . Y., 1961, Chapter 5.
D. H. RAMMLER AND H. G. KHORANA
1998
ribonucleic acidss That the aminoacyl group is indeed carried by the terminal adenosine unit and that i t is involved in an ester linkage with the 2’- or 3’hydroxyl group has been shown by isolation and
$.IT!?
v I
6~i > ~
characterization of aIriiuoacy1adenosine after pancreatic ribonuclease degradation of CI4-labeled aminoacyl ribonucleic acid^.^^^ Detailed studies of the equilibrium constants of the enzymatic reactionssbf8-l0 leading to the aminoacyl ribonucleic acid formation show these reactions to be readily reversible. The synthetic work on aminoacyl ribonucleosides described in this paper was performed in 1959 as a part of our interest in the general chemistry of the amino acid acceptor ribonucleic acids and particularly in developing approaches for separation of the individual amino acid acceptor ribonucleic acids utilizing the aminoacyl group as the handle. Furthermore, it was hoped that the availability of synthetic aminoacyl ribonucleosides and their protected derivatives would facilitate the study of the properties of this new class of compounds” and that this would in turn lead to a decision on the exact placement (2’- or 3‘-hydroxyl group in 11) of the a-a,minoacyl groups in the enzymatic reactions between aminoacyladenylates (I) and the terminal adenosine of the amino acid acceptor ribonucleic acids. The present paper describes a rather general method for the synthesis of 2’( or 3’)-O-aminoacyl ribonucleoside^.^^^^^ The facile isomerization (2‘0- e 3’-0) of the carbobenzyloxyphenylalanyl group in 5’-O-trityladenosine derivatives has been clearly demonstrated and it is concluded that interconversion of this type would Iikewise occur with an unprotected aminoacyl group, thus complicating the problem of determination of the location of such groups in the enzymatic reaction leading to 11. Finally, the probable ( 5 ) (a) E’. C. Zanircnik, “Harvey Lectures,” Vol. LIV, 250 (1958-1959); !b) F. Liymann, W .C. Hiilsmann, G. Hartmann, H. G. Boman and G. Acs, J . Crllular C‘omp. Physiol.. 54, 75 (1959); (c) P. Berg, A ? w Rev. Biochem., SO, 293 (1961). (6) H. G. Zachau. G. Acs and F. Lipmann, Proc. X d . A L Q ~ Sci. . U .S., 44, 885 (1958). (7) J. Preiss, P. Berg, E. J . Ofengand, F. H. Berymann and M. L k c k m a n u , ibid., 46, 319 (1959). (8)P. Berg and E. J. Ofengand, ibzd., 44, 78 (1Y58); P.Berg,,F. H. Berinan, E. J. Ofengand and M . Dieckmann, J . B i d . Chem., 186, 1728 (1961) (9) R . S. Schweet, F. C. Bound, E. Allen and E. Glassman, Proc. Ndl. A c a d . Sci. U . S., 44, 173 (1958). (10) I. D. Raake, Biochim. Biophys. Acta, 17, 418 (1958). (11) The presence of amino acids in ester linkages with hydroxyl groups of carbohydrate moieties of naturally occurring polymers appears t o be of more general significance. Thus d-alanine widely occurs in ester linkage with ribitol or glycerol hydroxyl groups in teichoic acids and glycerol phosphate polymers: See, e. g., J. Baddiley, J. G. Buchanan, R . 0. Martin and U. L. KajBhandary, Biochem. J . , 85, 49 (1962). (12) A preliminary report was published in 1900: D. H. Rammler and H. G. Khorana, Federation Proc., 19, 349 (1960). (13) Using a different method, other workers have since recorded the synthesis of 2’(or 3’)-0-leucyladenosinee and 2’(or 3’)-O-valyl-ribonucleosides [H. G. Zachau, Chem. Ber., 98, 1822 (19GO)I.
VOl. 85
reasons for the high lability of the aminoacyl ester of the nucleoside linkage are considered. Experiments were first carried out with a pyrimidine ribonucleoside, uridine, and the derivative chosen was one that carried a suitable protecting group in the 5’position. The steps involved are shown in Chart I. Carbobenzyloxy-DL-phenylalanine was converted, by reaction with dicyclohexylcarbodiimide, in excellent yield to the corresponding anhydridel4,l5 I11 which was isolated in a crystalline form and characterized by elemental analysis, infrared spectrum and conversion to the previously described anilide. Compound I11 served as the acylating agent for Ei’-O-tri-p-rnethoxytrityluridinel‘j (IV) under basic catalysis provided by pyridine. The total mixture (V-VII) was treated briefly with aqueous acetic acidls a t room temperature to remove the tri-p-methoxytrityl group and the resulting products were separated by partition chromatography on a silicic acid column. Carbobenzyloxy-DLphenylalanyluridine, presumably a mixture of 2’- and 3’-isomers (VI1 and VIII), was characterized by elemental analysis and negative periodate test. So long as the carbobenzyloxy protecting group was present on the a-amino group, the product was stable to storage in a solid state, there being no evidence of decomposition by paper chromatography. The same was the case in the adenosine analogs described below. The removal of the carbobenzyloxy group from VI1 and VI11 was accomplished by palladium-catalyzed hydrogenolysis in acetic acid a t low temperature and the product, 2’(or 3’)-0-(~~-phenylalanyl)-uridine (IX), was pure as determined by paper chromatography and paper electrophoresis. It was further characterized by quantitative spectrophotometric (for uridine) and ninhydrin (for phenylalanine) analysis. The starting material for the corresponding adenosine esters was 5’-0-trityladeno~ine~~,~~~ (TV, R = adenine, R’ = H). Reaction with the anhydride I11 as described above for the uridine derivatives gave the expected number of products, all of which could be separated (Fig. l) by chromatography on a silicic acid column. Thus, peak I contained unreacted carbobenzyloxy - DL - phenylalanine; peak 11, 2’,3‘ - di - 0(carbobenzyloxy - DL - phenylalanyl) - 5’ - 0 - trityladenosine ; peak 111, 3’-O-(carbobenzyloxy-~~-phenylalanyl)-5’-O-trityladenosine; peak IV, the 2’-O-isomer of the preceding compound; and peak V contained a small amount of unreacted 5‘-O-trityladenosine. Closer examination of the products in peaks I11 and IV showed their facile interconversion. Thus when a mixture of peaks I1 and I11 (peak I1 served as the reference) was rechromatographed on a fresh column of silicic acid, some of the material corresponding to peak IV was formed a t the expense of peak I11 (Fig. 2 ) . When purified peak IV, the amount of which normally (14) (a) H . G. Khorana, Chem. Rev., 58, 145 ( 1 9 5 3 ) ; (b) ref. 4, Chapter 6. (15) Other workers have independently reported on t h e preparation of symmetrical anhydrides of N-acylamino acids by t h e carbodiimide method. 1 . Muramatsu and A. Hagitani, J. Chem. SOL.Japan, Pure Chem. Section (Nippon Kosaku Zassi), 80, 1497 (1959); H. Schussler and H. Zahn, Chem. Ber., 95, 1076 (1962). (16) M. Smith, D. H. Rammler, I. H. Goldberg and H. G. Khorana, J . A m . Chem. Soc., 84, 430 (1962). (17) While the use of t h e ti’-O-tri-p-methoxytrityl group in t h e uridine series enabled t h e very facile removal of this protecting group, the use of t h e more stable trityl derivative in the adenosine series made possible the repeated chromatography o n silicic acid columns of the protected phenylalanine esters without the loss of the trityl group, This in turn enabled the orientation of t h e esterifying group on t h e 2’- or t h e 3’-hydroxyl by the phosphorylation of t h e sole remaining hydroxyl in the adenosine moiety and ready identification of adenosine-2’ or -3’ phosphate. (17a) NOTE ADDEDIN PROOF.-We have recently become aware of the independent synthesis of 2’(or 3’) -O-carbobenayloxyphenylalanyl-5’-0-trityladenosine described in Russian literature: E. Ya. Dreiman, V. A. Dmitrieva, S. G. Kamzolova, Z. A. Shabarova and M. A. Prokof‘ev, Zh. Obshch. Khim., 31, 3899 (lY61); Chem. Abstr., 57, 9936 (1962).
July 5, 1963 CHART
I.
SYNTHESIS OF 2’(OR 3’)-0-(DL-PHENYLALANYL)-RIBONUCLEOSIDES
BC-N-C&O HH
OH OH IV, R =uracil or adenine R’= OCHs or H
+ V Hfor iHBr an V I acetic Acid
1999
2 ’ ( 0 R 3’)-0-(DL-PHENYLALANYL)-RIBONUCLEOSIDES
~ H ~ c ~ H ~ v + 2’,3‘-di-O-hmer
VI1
b OH + H I H~N-C-C=O I CHzCsHa IX
+
2’- 0-isomers
c
VI
0
was less than that of peak 111, was rechromatographed, both peaks 111 and I V were again obtained (Fig. 3)) the two products being present in about equal amounts. The interconversion appeared to be catalyzed by the acidity provided by the column and/or by the solvent.’* Under the conditions used the conversion of peak I V to I11 (3’-O-~arbobenzyloxy-~~-pheny~a~any~ ester) appeared to be faster than the reverse process. (This migration was found to occur also under basic catalysis as shown below.) Phosphorylation of peak I11 in pyridine with a mixture of @-cyanoethylphosphate and dicyclohexylcarbodiimidelS and subsequent removal of the protecting groups gave mainly (85%) adenosine-2’ phosphate. 2o Phosphorylation of the isolated peak IV gave adenosine3’ and -2’ phosphates in about equal amounts and i t seems very probable that in this case base-catalyzed migration occurred in favor of the 3‘-O-isomer. From Fig. 1.-Products of the reaction of 5’-0-trityladenosine with the total of these results the conclusions are drawn carbobenzyloxy-DL-phenylalanyl anhydride; chromatography that peak I11 contained 3‘-O-(carbobenzyloxy-~~- on a silicic acid column; conditions as described in text: peak I, phenylalanyl)-5’-0-trityladenosine(IV, R = adenine, carbobenzyloxy-Dbphenylalanine; peak 11, 2’,3’-di-O-(carboR’ = H) and peak IV the isomeric 2’-O-ester. Interbenzyloxy-~~-phenylalanyl)-5’-0-trityladenos~ne; peak 111, 3’conversions are facile and are probably catalyzed both 0-(carbobenzyloxy-~~-phenylalanyl)-5’-0-trityladenosine; peak by acid22and base.21 IV, the S’-O-isomer of the preceding compound; peak V, 5’-0(18) I n one experiment, purified peak 111 was heated in reagent grade chloroform for 1 hr. and t h e product rechromatographed under t h e standard conditions. Both peaks 111 and IV were present. (19) G. M. Tener, J. A m . Chem. Soc., 8 3 , 159 (1961). (20) During t h e alkaline treatment t o remove t h e carbobenzyloxyphenylalanyl group and t o eliminate t h e 8-cyanoethyl group, t h e former must have come offfirst and t h e possibility of some transesterification t o form t h e 2’,3’cyclic phosphate‘ while t h e cyanoethyl group was still present cannot be ruled out. I n fact i t could have contributed t o t h e small extent of randomization of t h e phosphate group t h a t was encountered in this experiment. T h e other possibility for t h e presence of some 3’-phosphate is t h e acidcatalyzed isomerization before t h e phosphorylation actually took place. (21) T h e results are consistent with those recorded previously [D. H. Rammler and H. G. Khorana, J . A m . Chem. Soc., 84, 3112 (1962)) on the phosphorylation of N,O~’,O5’’-tribenzoyl-and N,02’,05’-tribenzoylcytidines. T h e former gave only cytidine-2’ phosphate while t h e latter gave, in addition t o the 3’-phosphate, some of the 2’-phosphate. There, t h e migrations were probably base-catalyzed and occurred in favor of t h e 3’-O-benzoyl derivatives. (22) Acyl group migrations under acidic conditions have been demonstrated by a large number of investigators in t h e carbohydrate and other fields. Only a few references are given here. A. Doerschuk, J. A m . Chem. Soc., 74, 4202 (1952); R. K. Ness and H. G. Fletcher, J. Org.Chem., 41, 1470 (19.57); Lohnizen and P. E. Verkade, Rec. frau. chim., 79, 133 (1960); R. C. Hockett, J . A m . Chem. Sac., 68, 928 (1946); E. E. van Tamelen, ibid., 7 3 , 5773 (19.51).
trityladenosine.
50,
(IO
Fig. 2.-Rechromatography of combined peaks I1 and 111 of Fig. 1 on a silicic acid column; note the appearance of peak IV.
Brief treatment of 3’-O-(carbobenzyloxy-~~-phenylalanyl)-5’-O-trityladenosinewith hydrogen bromide in acetic acid removed the trityl group and the product (VI1 VIII, R = adenine) was purified b y chromatography on a silicic acid column and characterized by
+
D. H. RAMMLER AND H. G. KHORANA
2uoo 36
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Vol. 85
investigation into model systems25and a combination of inductive effects, steric effects and hydrogen bond formation with the neighboring hydroxyl group has been invoked as possible explanations for the high rate of hydrolysis. As pointed out earlier,26a major factor responsible for the high lability of the aminoacyl ester linkage in ribonucleosides is the presence of the free neighboring hydroxyl group. Marked labilization of ester linkage in monoesters of cis-diols has been demonstrated by a number of workers.27 Hydrogen bonding as in Xz7lz8 or as in XI25bcould facilitate the approach of a hydroxyl ion or a water molecule and cause stabilization of the H O C U
H
C
I
Fig. 3.-Partial conversion of purified peak I V into peak 111; chromatography on a standard silicic acid column.
paper chromatography and by elemental analysis. Removal of the carbobenzyloxy group was accomplished by hydrogenolysis and 2'(or 3')-O-(~~-phenylalanyl)adenosine was pure as determined by paper electrophoresis a t acid pH. Full characterization was accomplished as described above for the uridine analog. In contrast with the stability of the carbobenzyloxym-phenylalanyl esters, the free aminoacyl esters showed high lability. The rate of hydrolysis was followed in 0.1 M phosphate buffer a t 2.5' and 34' by following the release of periodate-sensitive nucleoside. The half-life of DL-phenylalanyl ester of adenosine was 48 min. a t 25' and 22 min. a t 34'. The reaction followed first-order kinetics for the major part (up to about 70-80%) of the hydrolysis, but some deviation from this order occurred in the remaining part. The results are in general agreement with those of a number of previous ~ ~ r k e r ~in ~that ~ they . ~ show - ~ ~the. ease ~ ~ of hydrolysis of the aminoacyl ester linkage. Discussion.-The method chosen for the synthesis of the amiiioacyl esters is general as demonstrated for a pyrimidine and a purine ribonucleoside. The amino group in the adenine ring caused no interference in selective acylation a t the 2'- or 3'-hydroxyl groups with the protected amino acid anhydride. The results are consistent with the extensive studies reported from this Laboratory on the rates of acetylation of the hydroxyl groups and the amino group in adenine nucleosides. The method herein described is simpler and gives much higher yields than those previously reported. Clear demonstration has been given of the facile migration of carbobenzyloxy-DL-phenylalanylgroup between the 2'- and 3'-hydroxyl functions under acidic catalysis. The results are consistent with our own previous findings in the cytidine series and indeed with a large body of earlier work in the carbohydrate field.22 Although we have not attempted to demonstrate similar migration in the unprotected aminoacyl esters, it is our conclusion that the migration there would be even more rapid, being facilitated a t acidic or neutral PH by the inductive effect of the protonated a-amino group.23 This complication would apply directly to the determination of the position of the aminoacyl group in the enzymatic formation of aminoacyl ribonucleic acids. A large body of data is now available which shows the rather striking lability of the aminoacyl ester linkage.24 The finding has prompted a great deal of (23) Henry (24) sine-5'
O
a
,
E . S. Gould, "Mechanism and Structure in Organic Chemistry," Holt and Co.. New York, N . Y . , 1959, p. 207. The equilibrium constants of t h e over-all reactions involving adenotriphosphate, a-amino acids and amino acid acceptor ribonucleic
I
'0:
R X
XI
transition state associated with the attacking species. Labilization of the amino ester linkage must also be caused by the inductive effect of the protonated aamino g r o ~ p . ~(It~ ,is~ highly ~ probable that the species undergoing hydrolysis is that in which the aamino group is protonated.) Direct support for this conclusion was provided in the present work by the finding that the ester linkage was much more stable while carbobenzyloxy group protected the a-amino group. 30
Experimental3 General Methods.-Reagent grade pyridine dried over calcium hydride was used. Evaporations were carried out in vacuo a t below 40' bath temp. Silicic acid chromatography was performed using Mallinckrodt analytical grade silicic acid (100 mesh). The standard conditions used were: A column of 2 cm. dia. and containing 30 g. of silicic acid was used. Elution was performed using a linear gradient of a polar solvent, methyl alcohol in chloroform or ether. The mixing vessel contained 1 1. of pure chloroform (or ether) and the reservoir contained 1 1. of chloroform plus 5-1070 niethyl alcohol as specified in individual experiments. A flow rate of about 1 mi. per minute was maintained by application of a slight positive pressure of nitrogen. Periodate oxidations were followed by the spectrophotometric method described e l s e ~ h e r e . Quantitative ~~ amino acid analyses were performed using the ninhydrin method. 33 Paper chromatography was performed by the descending technique using Whatman No. 40 (double acid-washed) paper. Nucleosides and related compounds were detected by viewing under an ultraviolet lamp; amino acid esters were detected by acids"8-'0 show ready reversibility. Indeed, t h e result is a s would be expected on t h e basis of the value previously established for t h e free energy of hydrolysis of a simple carboxylic ester, ethyl acetate [F. H . Carpenter, J . A m . Ckem. Soc., 82, 1111 (1960)I. (25) ( a ) H . G . Zachau and W. Karau, Ckem. Ber., 93, 1830 (1900); (b) T. Bruice and T . H . Fife, J . A m , Chem. SOC.,84, 1973 (1962), and the references cited therein; (c) Z. A. Shabarova, N . A. Hughes and J. Baddiley, Biochem. J.,83, 216 (1962). (26) H . G . Khorana in J. Cell. Comp. Pkysiol., 64, Suppl. 1, p. 85 (1959). (27) H. B. Henbest and B. J . Lovell, J . Chem. SOC.,19ti5 (1957); S. M. Kupchan and W. S . Johnson, J . A m . C'kem. SOC.,78, 3804 (19,513); see also S. M . Kupchan, S.K . Eriksen and M . Friedman, i b i d . , 84, 4159 (lY02). (28) Hydrogen bonding in cyclopentane-cis-1,2-diolhas been demonstrated by L. K u h n , i b i d . , 74, 2492 (1952). (29) T h e same effect causes dramatic lowering of t h e p K , of t h e carboxyl group in glycine and other a-amino acids, whereas acylation of t h e amino group returns t h e 0Ka's of t h e carboxyl groups t o t h e normal range: E . J . Cohen and J. T. Edsall, "Proteins, Amino Acids and Peptides," Reinhold Publishing Corp., Kew York, N. Y., 1943, p. 116. (30) See also K . Moldave, P. Castelfrianco a n d A. Meister [ J . Bioi. Chem., 284, 841 (1959) 1, who noted similar effects in the mixed anhydrides of a-amino acids and adenosine-5' phosphate (31) All melting points are uncorrected and elemental analyses were performed by W. Manser, Zurich, Switzerland. (32) D. H. Rammler and J. C. Rabinowitz, Anal. Biochem., 4, 116 (1902). (33) E . W. Yemm and E . C . Cocking, Anolysf, 80, 210 (1955).
July 5 , 1903
2' (OR ~')-O-(DL-PHENYLALANYL)-RIBONUCLEOSIDES
2001
ence of phenylalanine amide was confirmed by paper chromaspraying the chromatograins with ninhydrin or the hydroxamate tography (solvent A) using an authentic sample as marker. reagent . 3 4 Paper electrophoresis was performed in an apparatus Isomeric ( 2 ' and J')-O-Carbobenzyloxy-~~-phenylalanyl-5'-0of the type described by Markham and Smith.% The medium trity1adenosines.-To a solution of 5'-O-trityladenosine (0.256 used most frequently was 1 M acetic acid solution. The average g., 0.5 mmole) in anhydrous pyridine ( 7 ml.) was added carbopotential was 1 5 2 0 volts/cm. benzyloxy-DL-phenylalanine anhydride (0.435 g., 0.75 mmole). The solvent systems used for paper chromatography were: The reaction mixture was kept a t room temperature for 18 hr. n-butyl alcohol-ethyl alcohol-water (4:1: 5 , solvent A); n-butyl Pyridine was then removed by evaporation and the residue was alcohol-acetic acid-water (4:1 :5, solvent B ) ; isopropyl alcoholdissolved in a small amount of benzene. This solution was chroconcd, ammonia-water (7: 1:2, solvent C); isopropyl alcoholmatographed on silicic acid under the standard conditions, using concd. ammonia-0.1 M boric acid ( 7 :1:2, solvent D ) ; saturated a gradient of 10% methyl alcohol in anhydrous ether. The eluaqueous ammonium sulfate-1 iM sodium acetate-isopropyl alcotion results (Fig. 1) were: peak I, carbobenzyloxy-DL-phenylhol (80:18:2, solvent E). alanine, 0.168 g., fractions 2-4; peak 11,2',3'-di-O-carbobenzylCarbobenzyloxy-DL-phenylalanine Anhydride.-To an anhpoxy-DL-phenylalanyl-5'-O-trityladenosine,0.150 g., 28%. fracdrous ether sohiion (3 ml.) of carbobenzyloxy-DL-phenylalanine tions 5-6; peak 111, 3'-O-carbobenzyloxy-~~-phenylalanyl-5'(0.328 g., 1.1 nimoles) was added DCC (0.124 g., 0.6 mmole). 0-trityladenosine, 0.198 g., 5OYc,fractions 7-10; peak IV, 2'-0The symmetrical anhydride as well as dicyclohexylurea precipicarbobenzyloxy-~~-phenylalanyl-5'-O-trityladenosine, 0.04 g., tated from the solution immediately. After being kept a t room lo",:, fractions 17-22; peak i', 5'-O-trityladenosine, 0.027 g., temperature for 1 hour, the reaction mixture was cooled t o 5" lO(jL, fractions 32-36. and the precipitate was collected by filtration. The anhydride The compounds in the different peaks were characterized as: wab extracted with cold dry ethyl acetate (10 ml.) and filtered 2 ',3 '-Di-O-carbobenzyloxy-~~-phenylalanyl-5 '-9-trityladenofrom the insoluble dicyclohexylurea. The solution was made opalescent with light petroleum ether (b.p. 30-40'). Alter 18 sine (Peak 11, Fig. l).-Hydrogenolysis of the carbobenzyloxy groups followed by alkaline hydrolysis gave adenosine and phenylhours at .5", this solution deposited needle-like crystals of the alanine as judged by paper chromatography in solvent B. The , n1.p. 125126'). .4 second crop of anhydride (0.21 g., crystals (rt1.p. 12%") was obtained from the inother liquor. The analytical sample was obtained by crystallization from aqueous 25C mfi in ethanol; m.p. 118-122", with shrinking a t 109'; , , ,A total yield wa5 905;. The infrared spectrum of the product methyl alcohol. shoffed two carbonyl absorption bands (1132 c m - ' strong, 1752 cm:-' niediurn) characteristic of carboxylic acid anhydride^.^^ Anal. Calcd. for C63H~i010'Ui(1072 41): C, 70.6; H, 5.37; .4nul. Calcd. for CpgH,j?OiSr(580.62): C, 70.3; H , 5.56; S , S , 9.16. Found: C, 69.88; H, 5.35; N,9.52. 4.83. Found: C, 69.49; H , 5.56; N,5.10. 3 '-O-Carbobenzyloxy-~~-pheny1alanyl-5 '-0-trityladenosine Carbobenzyloxy-DL-phenylalanine anilide (0.U1 g.) was ob(Peak 111, Fig. l).-A small amount of the material (0.005 9.) was heated in a boiling water-bath for 30 minutes in 80% aqueous tained by treating the above anhydride (0.017 9.) with freshly acetic acid ( 5 ml,). The cooled solution was hydrogenated with distilled aniline (0.03 nil.) in dry toluene ( 2 ml.); n1.p. 156-159" palladium-on-barium sulfate as described above. After 2 hr., quote m.p. 159-160'). (Anderson, et 2 '(or 3 ' )-0-Carbobenzyloxy-n~,-phenylalanyluridine .-To a water (2 ml.) was added, and the precipitated triphenylcarbinol solution of 5'-O-tri-p-methoxvtrity!uridine (0.1 g., 0.17 nimole) in and the catalyst were removed by filtration through a Celite pad. freshly distilled anhydrous tetrahydrofuran (3 nil.) was added The filtrate nas evaporated t o dryness and the residue used for anhydrous pyridine (0.03 nil.) followed by carbobenzyloxy-DLquantitative determinations. The phenylalanine t o adenosine phenylalanine anhydride (0.125 g., 0.22 mmole). The mixture ratio was found to be 0.97. An analytical sample was prepared was kept for 18 hr. a t rooin temperature with the exclusion of by freeze-drying a benzene solution of the peak material. The (ethyl alcohol) at 259 mp, the spectrum being sample had ,,,A, moisture. After this time, a chip of ice was added to destroy any residual anhydride and the solution was evaporated t o dryness. very similar to that of adenosine. The equivalent weight as The residue was dissolved in 805% aqueous acetic acid (3 ml.) and based on the adenosine chromophore was found t o be 775. after 15 min. at rootii temperature the acetic acid was evaporated. Anal. Calcd. for C&&Ss (790.85): c, 69.9; 13, 5.35; N, .\fter compiete removal of the solvent, the residue was dissolved 10.63. Found: C,69.34; H, 5.44; N, 10.53. in a small amount of benzene and the insoluble uridine (about 1%') Phosphorylation of 3'-O-Carbobenzyloxy-~~-phenylalanyl-5'was removed by filtration. The clear benzene solution (about 5 0-trityladenosine (Peak 111, Fig. 1) with p-Cyanoethyl Phosphate ml. ) was then passed onto a standard silicic acid colunin and elution and DCC.-To an anhydrous pyridine solution (0.5 ml.) of the l15-ni1. fractions collected) was carried out as follows: A mixture material in peak 111 (0,010 9.) and pyridinium p-cyanoethyl of anhydrous ether (75%) and benzene (25%) eluted tri-pphosphate (0.05 mmole) was added DCC (0.05 g.) and the sealed rnethoxytritanol in the first three fractions and carbobenzyloxymixture kept a t room temp. After 3 days, water (0.5 ml.) was phenylalanine in the next three fractions. Elution was then conadded and the solution kept a t room temperature for 12 hr. The tinued using standard conditions with a linear gradient of 10%) solution then was evaporated to dryness several times with small methyl alcohol in chloroform. The first nucleoside-containing peak eluted \%-as 2',3'-di-O-carbobenzyloxy-DL-phenylalanyluri- amounts of ethanol to ensure complete removal of pyridine. The dry residue was dissolved in chloroform (10 ml.) and hydrodine (11.2(,,;) and it was followed by 2'(or 3')-O-carbobenzyloxybromic acid in acetic acid (0.1 ml., 0.380 mmole) was added. DL-phenylalanvluridine (80c,c). This product was obtained as a After 5 minutes at room temperature, the solution was evaporated fine white powder by freeze-drying from a benzene solution cont o dryness and the residue was dissolved in a small amount of ditaining a small amount of methyl alcohol. oxane (1 d.).To this solution were added 9 ml. of concd. amA n d . Calcd. for C.'tiH.'YOVS,j (557.54): C, 58.3: H, 5.62; monia. The solution was heated at 50' for 3 hr. and then S , 7.53. Found: C, 58.29; H, 5.65; N, 7.29. evaporated t o dryness. The residue was dissolved in a small 2'( or 3')-O-~~-Phenylalanyluridine.-Carbobenzyloxy-~~- amount of water and the solution filtered from the insoluble tritanol phenylalanyluridine (0.05 g.) was hydrogenated in ice-cold 80% and dicyclohexylurea. The total solution was chromatographed in aqueous acetic acid ( 0 nil.) using palladium-on-barium sulfate solvent C. The products were found to be adenosine and the (0.05 g.) as the catalyst.38 Hydrogenolgsis zf the carbobenzylcorresponding nucleotide. The nucleotidic band was cut out and oxy group was complete in 90 minutes a t (1 , and the catalvst rechromatographed in solvent E. The major nucleotidic comwas removed by filtratioii through a sniall Celite bed. The cold ponent (85%) was identified as adenosine-2' phosphate and the clear filtrate was examined as follows: An aliquot was subjected minor component (15%) had an Rr identical with that of adenoto paper electrophoresis in 1 Af acetic acid. Only one ultravioletsine-3' phosphate. absorbing band was detected. This band moved under the condi2 '-0-Carbobenzyloxy-DL-phenylalanyl-5 '-0-trityladenosins tions used toward the cathode a t a rate of about 9 cm./hr., show(Peak IV, Fig. l).-The analysis was performed on a sample obing a net positive charge. The band gave positive reactions with tained by freeze-drying the benzene solution of the material in ninhyrin and hpdroxylarnine sprays. Quantitative ninhydrin this peak. The ultraviolet absorption characteristics were similar analysis and ultraviolet absorption measurement performed on an t o those of adenosine. The equivalent weight as based on the exaliquot gave phenylalanine and uridine in the ratio 1. Another tinction of adenosine was 826. After phosphorvlation using exaliquot was diluted with water and freeze-dried t o yield a powder actly the conditions described above for peak I11 (Fig. 1) adenowhich was kept in 1 ml. of concd. ammonia. After 2 hr. a t O 0 , sine-3' phosphate and adenosine-2' phosphate were obtained in the ammonia was removed by evaporation, and the residue about equal amounts. examined by paper electrophoresis. Uridine, phenylalanine and Anal. Calcd. for CIBH~*O?X*;~ (790.85): C , 69.9; H, 5.35; phenylalanine amide were identified as the products. The presN, 10.63. Found: C, 69.42; H, 5.55; N, 10.52. Interconversion of Peaks 111and IV of Fig. 1.-Peaks I1 (2',3'( 3 4 ) F. I.i]>mann and L. C. 'l'uttle, J . Bioi. ('hem., 169, 21 (1945). di-0-carbobenzyloxy-~~-phenylalanyl-5'-O-tr~tyladenos~ne, 0.150 ( 3 5 ) R . Markham and J. 1). Smith, Biochem. J . , 62, ,5,52 (1Y52). g.) and 111 (3'-O-carbobenzyloxy-~~-phenylalanyl-5'-O-tritylad(36) I,. J. Bellamy, "The Infra-Red Spectra of Complex Molecules," enosine, 0.198 g.) from Fig. 1were combined and the total dissolved J Wiley and Sons, Inc., Xew York, N. Y . , 1958. in a small amount of benzene. The solution was applied to the (:37) G W. Anderson, J. Blodinger, R. W. Young and A . 1 ) . Welcher, top of a standard silicic acid column. (In this column, peak I1 J . A l n . C h m . .So
