[15] Sugar nucleotide synthesis by the phosphoromorpholidate procedure

136

PREPARATION OF SUBSTRATES

[15]

the thiobarbituric acid-reactive material. Further, the product is fully reactive (in the presence of CTP) with CMP-3-deoxyoctulosonate synthetase to form CMP-3-deoxyoctulosonate-l~C. All the radioactivity is present in C-1 of the compound as evidenced by the loss of all of the radioactivity when the compound is decarboxylated with ceric sulfate. ~ 4E. C. Heath and M. A. Ghalambor, Biochem. Biophys. Res. Commun. 10, 340

(1963).

[15] Sugar Nucleotide Synthesis b y the Phosphoromorpholidate Procedure

By J. G. MOFFATT An extraordinary number of nucleoside diphosphate sugars with diverse biological functions are now known to occur naturally. Many of these compounds are present in extremely small amounts and their isolation in a pure form is laborious. Efficient general methods of chemical synthesis have, however, made it possible to prepare reasonable quantities of the natural coenzymes and of a variety of analogs. The most generally used synthetic method consists of the formation of the pyrophosphate bond in the coenzyme III through the reaction of various readily prepared and stable nucleoside 5'-phosphoramidates (I) with the appropriate sugar phosphate (II) in an anhydrous organic solvent according to Eq. (1). 1,2 N--POCHo/O~B

,o-C_/ OH

(1)

+ Rs~O--P--(OH)2--~-Rg-O - - P - - O - - P - - O C H 2 / O ~ B

OH

OH

(rf)

OH

(m)

Methods for the chemical synthesis of sugar phosphates are presented in this volume [11] and [12] as well as in Volume III [16A]. With the availability of these compounds the preparation of a wide variety of sugar nucleotides becomes straightforward. 1 j. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc. 80, 3756 (1958). S. Roseman, J. J. Distler, J. G. Moffatt, and H. G, Khorana, J. Am. Chem. Soc. 83, 659 (1961).

[15]

137

PHOSPHOROMORPHOLIDATE PROCEDURE

The Synthesis of Nucleoside 5'-Phosphoramidates The synthesis of a wide range of nucleoside 5'-phosphoramidates (I) is possible through the condensation of a nucleoside 5'-phosphate (IV) with ammonia, or with primary or secondary amines (V), in the presence of dicyclohexylcarbodiimide (VI) as in Eq. (2). 2'4

0 II 0 (HO)~P--OC~B

OH

C6HnNzC----N-C6Hn~-H~ ~ 0 + H~ -~ B ~/NH / N--P--OCH2/ I R~ (VI) Rz OH ~ ~ OH

(IV)

OH (v)

OH

(I)

The most widely used derivatives of type (I) are the nucleoside 5'-phosphoromorpholidates (VI) which have been chosen because they offer the best compromise between ease of formation, reactivity, and solubility in anhydrous organic solvents. The morpholidates of all the common ribo- and deoxyribonucleotides, and of a variety of nucleotide

--POCHz/v~

~)H~ OH

(w)

B

~C--N

O

CoH~,H/N %--/

OH

(vn)

analogs, have been prepared essentially according to the following general procedure? A solution of dicyclohexylcarbodiimide (824 mg, 4 millimoles) in tertbutyl alcohol (15 ml) is added dropwise over 2-3 hours to a refluxing solution of the nucleoside 5'-phosphate (1 millimole of the free acid or morpholine salt) in a mixture of water (10 ml), tert-butyl alcohol (10 ml) and distilled morpholine (0.34 ml, 5 millimoles). After a further 1-2 hours under reflux the mixture is directly examined by paper electrophoresis at pH 7.5 (0.05 M ammonium bicarbonate). The product moves with roughly one-half the mobility of the starting material and, if the reaction is not complete, further amounts (2 millimoles each) of morpholine and dicyclohexylcarbodiimide are added and refluxing is continued for a further hour. Complete conversion into the nucleoside phosphoromorpholidate is usually obtained under these conditions. The mixture is s R. W. Chambers and J. G. Moffatt, J. Am. Chem. Soc. 80, 3752 (1958). 4 j. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc. 83, 649 (1961).

138

PREPARATION OF SUBSTRATES

[15]

then cooled to room temperature and filtered, the crystalline dicyclohexylurea (m.p. 234 °) being washed with .tert-butyl alcohol and then water. The filtrate is evaporated in vacuo to roughly one-half its volume and then extracted twice with ether to remove excess dicyclohexylcarbodiimide. The clear aqueous solution (filtered if necessary) is evaporated to dryness in vacuo giving a glassy froth which is thoroughly dried on an oil pump. The residue is transferred in a minimum volume of methanol to a 40-ml centrifuge tube and carefully concentrated in vacuo to a volume of roughly 3-4 ml. Addition of dry ether (30 ml) precipitates a gum which, upon trituration with fresh ether, gives a dry, white powder. After a further wash with ether the product is immediately dried in vacuo at room temperature. The nucleoside 5'-phosphoromorpholidates prepared as above are obtained as salts of 4-morpholine N,N'-dicyclohexylcarboxamidine (VII) and tend to be hydrates. Crystalline products are generally not obtained, but the isolated yields of chromatographically and electrophoretically pure products usually exceed 90%. In view of the usual hydration of the products it is essential to determine the equivalent weight by ultraviolet spectroscopy using the same value for c.... as the parent nucleotide. It is also possible to prepare nucleoside 5'-phosphoramidates via activation of the nucleotide with other reagents such as carbonyl diimidazole 5,~ or diphenyl phosphorochloridate, ~ but these methods offer little advantage. Formation of the Pyrophosphate Bond

The reaction of the nucleoside phosphoramidate with a soluble amine salt of the sugar phosphate in a suitable anhydrous organic solvent leads directly, as in Eq. (1), to the formation of the pyrophosphate bond. The most frequently used solvent for this reaction is anhydrous pyridine (dried by distillation from, and storage over, calcium hydride), and, in general, the directly obtained 4-morpholine N,N'-dicyclohexylcarboxamidine salts of the nucleoside 5'-phosphoromorpholidates are soluble in this solvent. Guanosine 5'-phosphoromorpholidate has only a limited solubility in anhydrous pyridine but will generally dissolve upon addition of the sugar phosphate. Unsubstituted nucleoside 5'-phosphoramidates [(I), R1----R2----H] are much less soluble in pyridine and are accordingly less useful. The addition of o-chlorophenol has been successfully used to obtain homogeneous reactions, 1,8 and it is anticipated that F. Cramer and H. Neunhoeffer, Ber. 95, 1664 (1962). L. Goldman, J. W. Marsico, and G. W. Anderson, J. Am. Chem. Soc. @2, 2969 (1960). A. M. Michelson, Biochim. Biophys. Acta 91, 1 (1964). 8 M. Honjo, Y. Furukawa, and Y. Kanai, Biochim. Biophys. Acta 91, 525 (1964).

[15]

PHOSPHOROMORPHOLIDATE PROCEDURE

139

rigorously anhydrous dimethyl formamide or dimethyl sulfoxide could be similarly employed. Usually the sugar phosphates are allowed to react as their trialkylamine salts in order to obtain pyridine-soluble derivatives. Frequently it is necessary to use the tri-n-octylamine salts to achieve this goal. It is important that the reaction mixture be rigorously anhydrous during the condensation step. This is most readily accomplished by several evaporations to dryness i n v a c u o of pyridine solutions of the morpholidate and sugar phosphate. The reaction is generally complete within 1-2 days at room temperature but can be successfully carried out at 50-60 ° for a shorter period2 Purification of the products is conveniently accomplished by ion exchange or paper chromatography. The method is readily adaptable to microscale work as described in this volume [16]. A typical application of this method to the synthesis of uridine diphosphate glucose follows and may be directly extended to many related compounds. The Synthesis of Uridine Diphosphate Glucose ( U D P G ) 2 The 4-morpholine N,N'-dicyclohexylcarboxamidine salt of uridine 5'phosphoromorpholidate (0.33 millimole) is dissolved in anhydrous pyridine (10 ml) and evaporated to dryness i n v a c u o . This procedure is repeated twice with readmission of dried air. Separately an aqueous solution of dipotassium glucose-a-l-phosphate.H20 (1 millimole) is passed through a 1 X 5 cm column of Dowex 50 (pyridinium) resin and the eluate and washings are concentrated to a volume of 5 ml. Pyridine (15 ml) and tri-n-octylamine (1 millimole) are added and the clear solution is evaporated to dryness in v a c u o . The residue is rendered anhydrous by three evaporations with dry pyridine and added, in pyridine, to the morpholidate. The mixture is evaporated once more, dissolved in dry pyridine (5 ml), and stored at room temperature for 2 days. The solvent is then evaporated and the oily residue is suspended in water and stirred with lithium acetate (150 mg). Trioctylamine is then extracted with ether and the aqueous phase is applied to a 2 X 10 cm column of Dowex 2 resin in the chloride form. Following a water wash the products are eluted with a linear gradient of 4 1 of lithium chloride in 0.003 N hydrochloric acid (from 0.01 to 0.10M salt). Any unreacted morpholidate and uridine 5'-phosphate are eluted first, followed by U D P G (81% by ultraviolet absorption) at roughly 0.06M salt. The 9N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, G. I. Yeliseeva, M. A. Grachev, and V. P. Demushkin, Tetrahedron 19, 1207 (1963).

140

PREPARATION OF SUBSTRATES SUGAR NUCLEOTIDES PREPARED BY THE PHOSPHORAMIDATE METHOD

Nucleoside diphosphate sugar

Methods~'b and yields

UDP-glueose UDP-~-glucose UDP-galactose UDP-glucuronic acid UDP-~-glucuronic acid UDP-rhamnose UDP-xylose UDP-N-acetylglucosamine UDP-N-acetylgalactosamine 2oThio UDP-glucose 4-Thio UDP-glucose Na-Methyl UDP-glucose 6-Aza UDP-glucose 5-Bromo UDP-glucose 5-Bromo UDP-~-glucose Pseudo-UDP-glucuronic acid

(M, 70%), c (I, 67%), d (A, 59%). (A. 50%)/ (M,--)~ (M, 70%), c (M,--),g (M,--)~ (M, 66%), c (A, 58%)' (A, 44%)' (M, __)i (M, __)k (M, 68%). c (M. 65%) t (M, 75%). "~ (M, 64%)" (M, 50%) ° (M, 46%) ° (M, 40%) 0 (M, 50%). (h, --)i (h, _ ) i (h, 32%)p

ADP-glueose ADP-fl-glucose ADP-galactose ADP-mannose ADP-xylose ADP-maltose ADP-ribose-(5) ADP-glucose-(6) ADPoglyceric acid ADP-2-phosphoglyceric acid ADP-2-mercaptoethanol ADP-riboflavin(FAD) ADP-cobinamide 2-Deoxy-ADP-glucose 2-Deoxy-ADP-niboflavin

(M, __),o (M, __)k (M,--),~ (M, __)k (M, --)g (M, __)h (M, __)k (M, __),k (M,--)g (P, 25%) r (P, 37%)" (M, __)r (M. 30%) ° (A, 33%)* (I, 67%), ~ (A, 40%). (A, 18%),* (M, 24%)~ (M, _)k (M, --)~

GDP-mannose GDP-glucose GDP-fructose-(1) GDP-riboflavin 2-Deoxy-GDP-mannose 2-DeoxyoGDP-riboflavin CDP-glycerol CDPoribitol CDP-glucose CDP-mannose CDP-riboflavin 2-Deoxy CDP-riboflavin Iso-CDP-glucose

(M, 63%), c (C, 32%)* (C, 32%),* (M,--)~ 27(C, --)v 25(M, _)w 13(M, _)h 25(M, --)w (M, 70%), ~ (M, _),k (M,--)g (M, --)* (M, 40%), o (M, --),q (M, --)g (M, _),h (M,--)q (M, --) (M, --)= (M, 15%)°

TDP-glucose

(I, 70%), * (M, __),k (M,--)q

[15]

[15]

PHOSPHOROMORPHOLIDATE PROCEDURE

Nucleoside diphosphate sugar

141

Methods ~'b and yields

TDP-mannose TDP-galactose TDP-N-acetylglucosamine TDP-riboflavin

(A, 65%) ~" (M, __)h (M, __)bb (M, - - ) ~

IDP-glucose IDP-mannose

(M, - - ) ~c (M, __)h

a References to papers with no experimental detail are generally not given if a well documented synthesis has been described. References are cited in footnotes c-cc. b The type of nucleoside phosphoramidate used, and the yield obtained, are enclosed within parentheses using the following key: M, morpholidate; A, amide; C, cyclohexylamide; I, imidazolate; P, phenylalanine amide. c S. Roseman, J. J. Distler, J. G. Moffatt, and H. G. Khorana, J. Am. Chem. Soc. 83, 659 (1961). d F. Cramer, H. Neunhoeffer, K. H. Scheit, G. Schneider, and J. Tennigkeit, Angew. Chem. 74, 387 (1962). J. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc. 80, 3756 (1958). s T. Ueda, Chem. Pharm. Bull. (Tokyo) 8, 464 (1960). a E. Recondo and L. F. Leloir, Biochem. Biophys. Res. Commun. 6, 85 (1961). h j. Preiss and E. Wood, J. Biol. Chem. 239, 3119 (1964). M. Honjo, Y. Furukawa, K. Imai, H. Moriyama, and K. Tanaka, Chem. Pharm. Bull. (Tokyo) 10s 225 (1962). J G. A. Barber, Biochem. Biophys. Res. Commun. 8, 204 (1962). k M. Dankert, I. R. J. Goncalves, and E. Recondo, Biovhim. Biophys. Acta 81, 78 (1964). D. M. Carlson, A. L. Swanson, and S. Roseman, Biochemistry 3, 402 (1964). '~ J. E. Silbert, J. Biol. Chem. 239, 1310 (1964). n E. A. Davidson and R. W. Wheat, Biochim. Biophys. Acta 72, 112 (1963). o N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, G. I. Yeliseeva, M. A. Grachev, and V. P. Demushkin, Tetrahedron 19, 1207 (1963). M. Honjo, Y. Furukawa, and Y. Kanai, Biochim. Biophys. Acta 91, 525 (1964). q G. A. Barber, A. D. Elbein, and W. Z. Hassid, J. Biol. Chem. 239, 4056 (1964). TZ. A. Shabarova, T. S. Ryabova, and M. A. Prokofiev, Dokl. Acad. Nauk SSSR 136, 1116 (1961). T. Hashimoto, M. Tatibana, Y. Ishi, and H. Yoshikawa, J. Biochem. (Tokyo) 50, 548 (1961). * M. Ikehara, Chem. Pharm. Bull. (Tokyo) 8, 830 (1960). F. Cramer and H. Neunhoeffer, Ber. 95, 1664 (1962). K. Bernhauer and F. Wagner, Biochem. Z. 335, 453 (1962). w D. M. McCormick, B. M. Chassy, and J. C. M. Tsibris, Biochim. Biophys. Acta 89, 447 (1964). x T. Ueda and E. Ohtsuka, Chem. Pharm. Bull. (Tokyo) 7, 935 (1959). J. Baddiley, N. A. Hughes, and A. L. James, J. Chem. Soc. 1961, 2574 (1961). L. Glaser, J. Biol. Chem. 239, 3178 (1964). "~ N. L. Blumson and J. Baddiley, Biochem. J. 81, 114 (1961). bb S. Kornfeld, R. Kornfeld, E. F. Neufeld, and P. J. O'Brien, Proc. Natl. Acad. Sci. U.S. 52, 371 (1964). ~¢ H. Verachtert, S. T. Bass, and R. G. Hansen, Biochem. Biophys. Res. Commun. 15, 158 (1964).

142

PREPARATION OF SUBSTRATES

[16]

pooled peak is adjusted to pH 4 with lithium hydroxide and carefully evaporated to dryness in vacuo (bath temperature of 30°). The dry residue is dissolved in methanol (5 ml) and precipitated with acetone (30 ml) and ether (5 ml). The white precipitate is then reprecipitated several times from methanol with acetone and ether until the supernatant is free of chloride ions. The final yield of enzymatically active dilithium UDPG.6 H:O is 142 mg (70%). Li~UDPG.6 H20 requires: P, 9.02; P:glucose:uridine = 2.00:1.00:1.00 Found: P, 9.04; P: glucose: uridine ~-- 1.98: 0.98: 1.00 An almost identical procedure may be used for the synthesis of most simple sugar nucleotides of type III, and the table outlines many of the compounds that have been prepared by this method.

[ 1 6 ] M i c r o s c a l e A d a p t a t i o n of t h e M o r p h o l i d a t e P r o c e d u r e for t h e S y n t h e s i s of S u g a r N u c l e o t i d e s

By

ALAN D. ELBEIN

Principle. Sugar nucleotides can be prepared in large amounts in relatively good yields by the morpholidate procedure. 1 This method has also proved quite useful for preparing small quantities of radioactive sugar nucleotides when the amount of 14C-sugar 1-phosphate is limited. Furthermore, by elevating the temperature 2 it is possible to increase the speed of the reaction and thereby decrease the time of incubation (Fig. 1). The unreacted sugar 1-phosphate may be reisolated at the end of the reaction and recondensed, so that most of the sugar 1-phosphate may eventually be converted to sugar nucleotide. The procedure described here is essentially that described in [15] except that reactants are mixed in microamounts and the reaction is run at an elevated temperature. The preliminary isolation of the sugar nucleotide is then performed by paper electrophoresis rather than ionexchange column chromatography. Reagents

14C-sugar !-phosphate Nucleoside 5'-phosphoromorpholidate (nucleoside 5'-monophosphate morpholidate prepared as described in [15], or obtainable from California Foundation for Biochemical Research) S. Roseman, J. S. Distler, S. G. Moffatt, and H. G. Khorana, J. Am. Chem. Soc. 83, 659 (1961) ; see also this volume [15]. N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and M. A. Grachev, Biochim. Biophys. Acta 59, 747 (1962).