somal peptidyltransferase1'2. The activity of these ... - BioMedSearch

Nucleic Acids Research

Volume 4 Number 7 July 1977

Synthesis of acylamino acid esters of nucleoside 5'-phosphates and their investigation with PMR and CD spectra A. V. Azhayev, S. V. Popovkina, N.B. Tarussova, M.P. Kirpichnikov, V.L.Florentiev, A.A. Krayevsky, M. K. Kukhanova,ardd -.P. Gottlkh

Institute of Molecular Biology of the USSR Academy of Sciences, Vavilov str., 32,Moscow 117312, USSR

Received 14 April 1977

ABSTRACT The acylamino acid esters of nucleoside 5'-phosphates are synthesized via condensation of N-(N'-acylaminoacyl)imidazoles with nucleoside 5'-phosphates. The PM and CD spectra of the esters obtained are studied. The 3'-isomers of the substances under study are observed to have a shift in the conformational N*:S equilibrium of the carbobydrate moiety in favour of the S-form as compared to the initial nucleosides and their 2'-acyl esters. INTRODUCTION

Acylamino acid esters of adenosine and those of adenosine 5'-phosphate are widely used as model substrates of the ribosomal peptidyltransferase1'2. The activity of these analogs of the 3'-terminal fragments of peptidyl-tRNA and aminoacyltRNA is known to be strongly dependent on the nature of the amino acid residue. In connection with this, the spectral measurements estimating these compounds are of interest. The imidazole method of synthesis of nucleoside 5'-phosphate amino acid esters described by us earlier3'4 was developed for small scale preparations only with subsequent separation of products using paper chromatograpby. The present paper describes obtaining of large quantities of the above-mentioned IATIONS USED: A-adenosine; pA, pU, pG, pI and p0-51phosphates of adenosine, uridine, guanosine, inosine and cyti- 2' and 3'-0-metbyladenodine, cor espondingly; AM and A sines; pA and pAMe-5'-PhosphatIZ of ANa and AMe; A-Phe and pA-Phe-2'(3')-O-phenOlalanyl-adenosine and its 5'-phosphate; A-(flet) and pA- et)-21 (3 ')-O-(N-formylmethioxVl )-adenosine and its 5'-phosphates. pA-(fLeuGly)-2'(3').-O-(N-forylmethionylglycyl)adenosine 5'-phosphate. The esters of other amino acids, acylasino acids and acylpeptides with adenosine and all types of nucleotides were abbreviated analogously, pA-

iValer-22(3' )-O-iso-valeroyl-pA Boc -tert.-Butylo3qcarbonyl; -

CDI

N,N'--carbonyldiimidazole.

C> Information Retrieval Limited 1 Falconb.rg. Court London W1V5SFG England

2223

Nucleic Acids Research compounds. The study of the PIE and CD spectra of some of the above-mentioned esters has been made, too.

EYPERIKWTAL N-Forylamino acids were obtained from the I- and D.amino acids5. Boc-methionine was prepared from Boc-N and methioni3 ne as described6 . Method7 was applied for the synthesis of AMe and AMet and method8 for N-(acetoxsuccini±ide1Oxydation of pA into pAredred with NaJO4 and its subsequent reduction with PopoyainfAMe of A and AM NaBH4 was carried out as 4in9 i.. Phosphoylation was achieved with POC13 in (C2H50)3P0 solution b The TLC was performed on "Siluf ol UV2#" plates from Kavalier, Czechoslovakia in n-BuOH-AcOH-water (5:2:3 v/v, system Ml), and paper chromatograp1y on Whatman NI in n-BuOH.-AcOH-water (78:5:17 v/v, system 22). The PIl spectra were taken with a Tesla BS 487 spectrophotometer operating at 80 mHz, Chechoslovakia, in D20 with tert.-BuOH as internal standard. The CD spectra were observed with a Jobin-Ivon Dichrographe III. 2' (3' )--(N-Forml)-aminoaZl-nucleoside 5'-phosphates Method A. 5.5 moles of CDI was added to the solution of 5 mmoles of N-formql-L- or D-amino acid in 2 ml of DX!'. The mixture was stirred for 15 min until the C02 formation was stopped, and the solution obtained was added to 0.2 mmoles of nucleotide (disodium salt) in 2 ml of water (4 ml of water in the pC case). The reaction mixture was stirred for 2.5 hr at room temperature and diluted with 80 ml of cold acetone. After centrifugation the solid residue was washed with cold acetone and dried in vacua. The precipitate was dissolved in 1-2 ml of deionized water and applied to the silica gel coluimn 25x1.5 cm. Elution was made with system m1-° 2 at 40, 30 ml/hr. The fractions containing formylamino acid esters of nucleotides (control - TLC in system AL 1) were diluted with 2 volumes of ether, and the substances were extracted three times with 0.1 volume of water. The aqueous solution was shaken with ether and freeze-dried. The yields and certain characteristics of the obtained compounds are given in Table 1. Method B. N-(N'-For l)-aminoacyl-imidazole, prepared M4

Nucleic Acids Research from 2 mmoles of N-formlamino acid in 2 ml of DPU as described above, was added to the nucleotide (mono Na-salt) solution in 3 ml of dxr formamide. The reaction mixture was stirred overnight and then diluted with 50 ml of dxy acetone and 50 ml of ether. After centrifugation and washing with dzy ether, the isolation of compounds was carried out analogously to Method A. The above methods were also emplo7ed to obtain 2'(3')-0fonnyldipeptidyl-nucleoside 5'-phosphates via condensation of N-(N'-formyldipeptidyl)-imidazole and pA either in water /A/, or in organic solvents /B/.

2'(')-0U-Acetyl-L-

5'-p_hoshates (or_D)-methionl-adenosine

L or D isomers of Boc-methion7l-imidazole prepared from 2.2 moles of Boc-methionine and 2.e mmoles of CDI in 3 ml of DUN, were added to 2 mmoles of mono-Na-pA solution in 3 ml of formamide. The reaction mixture was stirred overnight at room temperature, and the compound obtained were precipitated with 35 ml of dry acetone and 35 ml of dry ether. The reaction mixture was centrifuged at 00, and the precipitate washed with dry ether and dried in vacua. The substances were dissolved in 5 ml of dry F3CCOOH at 00, the solution obtained was kept at room temperature for 20-25 min and evaporated in vacua. The residue was neutralized with Et? and added 3 mmoles of N-(acetov)-succinimide. The reaction mixture was stirred for three days, and the substances were isolated by Method B. The elemental analysis of all the esters synthesized revealed the deviation of experimentally obtained values from the theoretically calculated not to exceed 0.5%.

RESULTS AND DISCUSSION The synthesis of formVlaminoacyl- or formyldipeptidyl-nucleotides (V) is based on condensation of the corresponding acyl-imidazoles (III) with nucleoside 5'-phosphates (IV). The starting imidazoles (III) were obtained via the reaction of N-formqlamino acids or N-formyldipeptides (I) with CDI, Scheme 1. Since imidazoles (III) are readily bhdrolyzed in water, it is important in Method A to introduce the 2.5-fold molar excess {III) with respect to the initial nucleotide component. 2225

Nucleic Acids Research At the same time, in Method B the molar ratio of (III) to (IV) was 1:1,and the yields, as a rule, were better than those of Method A. Anyhow, Method B is not suitable for the preparation of esters of pC and pG (V). The initial pG failed to dissolve in formamide, and for pC, the main direction of reaction with (III) was acylation of the NH2-group of cytosine. The results of the synthesis of esters (V) are summarized in Table I. SCHIM 1

RNHCHRCOOH + ImCOIm I II

~RNCHIOOH29 IV~~~~

[I RHCMRCO-m JIII

~

2-ioe

where R-formyl or N-formylaminoacyl, B- adenine, guanine, cytosine, uracyl, hypoxanthine. The structure of components was demonstrated with routine procedures 3'4. The UV-spectra showed no base N-acylation in CV). The electrophoresis on paper,pH 6.4,testified to the presence of a monosubstituted phosphate group. The esters (V) could not be oxidized by NaJO4, but were readily hydrolized by 0.5 N NaOH in 2 min at 00 producing initial nucleotide and acylamino acid. The nucleotide-acylamino acid ratio was approx. 1:1 in moles. The imidazolide (III) formation was not accompanied with any noticeable racemization of N-formylmethionine (I) for both L or D-isomers which was demonstrated by a test such as this. After hydrolysis of the freshly prepared portions(100 mg each)of L- and D-isomers (III) in 0.1 N AcOH for 3 hr at 200, the mixture was evaporated and the residues dissolved in water and filtered through a dowex 50x4 (He, 200-400 MESH) column. The solutions obtained were evaporated. Table 2 represents [lD and the melting points of the initial (I) and those of the imidazolides (III) isolated after the hydrolysis. The racemization of 2226

Nucleic Acids Research the samples under test was no more than 5%. However after keeping it in DMF overnight the racemization of methionine in (III) reached 30%. Since protection of the amino acid NH2-group with other acyl groups - like an acetyl one, for example - could result in significant racemization, the synthesis of pA-(Ac-L-Met) and its D-isomer was made as is shown in Scheme 2. The reaction of Boc-L-methionine, or its D-isomer (VI) with (II)biSought about the imidazolide (VII). This, in compliance with the routine methods 394 was followed by a condesation with pA which gave rise to (VIII). After the reaction the protective group was removed from(VIII) using CF COOH 3'4 3 the methionyladenilic acids were put to the reaction with NTABLE 1 Yields and certain characteristics of acylaminoacyland peptidyl-nucleotides

Initial: |Compound

pA-(fMet)

pI-(fMet) pG-(flet) pC-(fM*t) pU-(fMet) pA-(fLeu) pA-(fPhe) pA-(fGly) pA-(fAsn) pA-(Ac-L-Met) pA-(Ac-D-Met). pA-(fLeuGly) pA-(fGlyLeu) pA-(fMetGly) pA-(fGlyMet)

pAM-(flet)

pA.U-(fMet) pAr.d-((fMet) PA

800 800 200 800 800

800 800 400 400 800 800 800 800 800 800 200 200 800

-

Seche

Yields

:nuoleo-: s tid : Method A: Method tidep % 8%

UV-

B:spect-

380 34 570 51 370 38 85 42.4 260 22.4 420 36.3 570 49.4 340 30.4 505 45.6 315 27.4 510 44.3 140 29.2 260 54.3 270 25.1 61 13.5* 40 9.6* 240 19.5 330 26.9 215 17.5 330 26.9 315 26.3 385 31.3 280 22.8 330 26.8 35 12.2 72 25.2 68 23.6 187 65.0 370 33.0 -

ra,,

Rf

~~~~insystem

NO11: NO02

259

0.35

262

0.38 0.12

255 272

265 259 259 259

959 259 259 259 259 259 259 259

259 259 259

0.27

0.33 0.46 0.40

0.40 0.28 0.30 0.39 0.39

0.43 0.42 0.41

0.41 0.48 0.52

0.10 0.07 0.10 0.15 0.10 0.12 0.03

0.03 0.15 0.15 0.12 0.12 0.11 0.11 -

0.38 0.12 0.02

2

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Nucleic Acids Research TABLE 2 Melting points and specific rotation (c I, water) of N-formyl-L-methionine and its D-isomer obtained via hydrolysis of thei3Z imidazolides

f-D-Met

f-L-Met

Initial elting points

[0]2O

Obtained from III

Initial

trom fo

iII I

98-1000

98-990

99-1000

98-1000

-9.80

_9,40

+10.00

+9.80

SCHEIY1E 2

BOC -Met VI ®o

+

[BOC-Met-Im I VII

ImCOlm

®OCH2

B

C

0