J. Mol. Evol. 10, 193--209 (1977)

Journal of Molecular Evolution © by Springer-Verlag 1977

Cyanamide Mediated Syntheses Under Plausible Primitive Earth Conditions II. The Polymerization of Deoxythymidine 5'-Triphosphate E. Sherwood, A. Joshi, and J. Orb* Departments of Biophysical Sciences and Chemistry, University of Houston, Houston,Texas 77004, USA

Summary. When an aqueous solution (pH 7.0) of 3H deoxythymidine 5'-triphosphate, deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide, cyanamide and ammonium chloride was dried and heated at 60°C for 18 h, oligomers were obtained in a yield of approximately 80%. After the chemical degradation of any pyrophosphate bonds present in these oligomers, linear polynucleotides of up to 7 - 8 units in length were isolated by DEAE cellulose column chromatography and identified by enzymatic digestion procedures. The di- and trinucleotide fractions were degraded 87% and 100% by snake venom phosphodiesterase and 39% and 9% by spleen phosphodiesterase. This synthesis of deoxythymidine oligonucleotides was conducted under potentially prebiotic conditions and may offer a possible method for the synthesis of deoxyoligonucleotides on the primitive Earth.

Key words: Prebiotic - Polymerization - Deoxythymidine oligonucleotidesDeoxythymidine 5'-triphosphate - Deoxythymidine 5'-monophosphate 4-amino-5-imidazolecarboxamide- Cyanamide

Introduction Cyanamide has been proposed as a plausible condensing agent for the polymerization of amino acids and nucleotides under primitive Earth conditions (Or6, 1963; Steinman et al., 1964). Under mild aqueous conditions it has been shown to promote the * To whom all correspondence should be addressed Abbreviations. The following abbreviations have been used in this paper; dT, deoxythymidine;

dTMP, deoxythymidine 5r-phosphate; dTppT,p 1, P27 dideoxythymidine 5r-pyrophosphate; dTTP, deoxythymidine 5 -triphosphate; "Pyr" dT, 5 -C-pyridinium deoxythymidine; "Pyr" dT(pT) n, 5'-C-pyridinium deoxythymidine oligonucleotides; AICA, 4-amino-5-imidazolecarboxamide

194

E.Sherwood et al.

condensation of amino acids to peptides, and nucleotides to oligonucleotides (Ponnamperuma and Peterson, 1965; Halman, 1968; Steinman et al., 1965a, b, 1966; Ibanez et al., 1971). The yields in most cases were low. Cyanamide was found to be more effective as a condensing agent when used under dehydrating conditions resulting from the drying of small ponds (Stephen-Sherwood et al., 1973; Odom et al., 1975; Schwartz, 1972). In the preceding paper (Sherwood and Or6, 1977) we reported that under dehydrating conditions cyanamide, in conjunction with 4-amino-5-imidazolecarboxamide (AICA), promoted the condensation of deoxythymidine 5'-phosphate (dTMP) to p1, p2_dideoxythymidine 5'-pyrophosphate (dTppT). In this paper we suggested that the condensation of deoxythymidine 5'-triphosphate (dTTP) on a dTppT primer, should yield linear oligonucleotides according to the following reaction scheme: ndTTP dTppT

~.. dTppT(pT) x + nPPi AICA + CNNH2

+ d(Tp)yTppT(pT) z

To test this hypothesis we dried and subsequently heated a solution containing dTTP, dTMP, AICA and cyanamide for 18 h at 60°C. Analysis of the products formed in this reaction indicated that linear oligonucleotides of up to 7 - 8 units in length were synthesized in an overall yield of 80%.

2. Experimental 2. i Materials

Cyanamide (mp 42°C) was purchased from Eastman Chemicals and 4-amino-5-imidazolecarboxamide hydrochloride (grade A) from Calbiochem. Deoxythymidine 5'phosphate disodium salt (Sigma) and deoxythymidine 5'-phosphate-2-14C diammonium salt (New England Nuclear), were checked in solvent systems I and lI (see below) and shown to be pure. Deoxythymidine 5'-triphosphate tetralithium salt (Schwarz) and deoxythymidine 5'-triphosphate-2-3 H tetralithium salt (Schwarz) were checked in solvent systems I and III and found to be contaminated with a small amount of deoxythymidine 5'-diphosphate. They were used without further purification. The standard oligonucleotide (pdT)2 was purchased from Collaborative Research. The dinucleotide dTpT was obtained from (pdT)2 by treatment with E. coli alkaline phosphatase. E. coli alkaline phosphatase (BAPF), snake venom phosphodiesterase and spleen phosphodiesterase were all purchased from Worthington. Thin layer polygram cel 300 PEI (polyethyleneimine) plastic sheets were obtained from Brinkman.

2.2 Chromatography and Electropboresis

Thin layer chromatography was carried out on PEI cellulose sheets using solvent system I. This system involved consecutive chromatography in two different solvents.

Cyanamide Mediated Syntheses II

195

The chromatogram was developed first in formic acid (20%), allowed to air dry overnight, and then developed again in the same direction in ammonium bicarbonate 2% (Stephen-Sherwood et al., 1973). Paper chromatography was performed on Whatman 1- and 3-MM paper by the descending technique in the following solvent systems. Solvent system II: isopropanol, ammonia, water (70, 10, 20; v/v) and solvent system III: n-butanol, acetic acid, water (50, 20, 30; v/v) (Weimann and Khorana, 1962). Paper electrophoresis was carried out on Whatman 3-MM paper using solvent system IV, 0.03 M phosphate buffer (pH 7.1), using a Savant flat plate electrophoresis apparatus at 3000 V for one hour. Column chromatography for the separation of the reaction products was done on a DEAE (diethylaminoethyl) cellulose column (HCO~form) according to the procedure of Khorana and Vizsolyi (1961). The DEAE cellulose column was 1.5 cm x 20 cm and the reaction products were eluted from the column using a 0.0-0.4 M triethylammonium bicarbonate (pH 7.5) gradient, contained in I liter mixing vessels. The flow rate of the column was approximately 6 ml in 18 min, and the effluent was analyzed using a Beckman DU spectrophotometer at 267 nm. All radioactivity measurements were made using a Packard liquid scintillation spectrophotometer Model 3380. Aquasol (New England Nuclear) was used for counting liquid samples, or samples eluted from paper chromatograms with pH 2 HC1 and toluene cocktail was used for counting strips from chromatograms.

2.3 Enzymatic Analysis The enzymatic analyses were performed according to the procedure given by Pongs and Ts'o (1969). For the removal of terminal phosphate groups from oligomers, 5/al of E. coli alkaline phosphatase (0.1 mg/ml) was added to 0.2 bl moles of nucleoside units in a totat volume of 0.035 ml of 0.5 M Tris buffer (pH 8.0). The mixture was incubated at 37°C for 4 h and analyzed by paper chromatography using solvent systems II or III. The enzyme was checked and standardized with dTMP. For the degradation of oligonucleotides by snake venom phosphodiesterase: 10/~1 of enzyme (5 mg/ml H2 O) was added to 0.2/a moles of nucleoside units in a total volume of 0.06 ml of 0.33 M NH4HCO 3 buffer (pH 9.0). The mixture was incubated at 37°C for 8 h and analyzed by paper chromatography using solvent systems II or III. The UV absorbing spots corresponding to dTMP and deoxythymidine were eluted from the paper with pH 2 HC1 and, where possible, the OD units per spot/ml at 270 nm determined. The enzyme preparation was checked and standardized with dTpT. Partial degradation of oligonucleotides with snake venom phosphodiesterase was performed as follows: 10/al of enzyme (0.5 mg/ml H2 O) was added to 0.2 # mole nucleoside units in a total volume of 0.06 ml 0.33 M NH4HCO 3 buffer (pH 9.0). The mixture was incubated at 37°C for 5 min at which time the reaction was terminated by heating in a boiling water bath for 1 rain. The products were analyzed by chromatography on PEI cellulose plates using solvent system I or paper using solvent system III. The TLC plates or paper were subsequently strip counted to determine the position of the radioactive components. The degradation of oligonucleotides by spleen phosphodiesterase was performed as follows: 20/al enzyme (16 units/ml water) was added to 0.2/~ mole nucleoside units

196

E.Sherwood et al.

in a total volume of 0.04 ml of 0.125 M sodium succinate (pH 6.5). After incubation at 37°C for 8 h, the products were analyzed by paper chromatography using solvent systems 11 or III. The enzyme was checked and standardized with dTpT.

2.4 Procedure A typical reaction consisted of an aqueous solution (8.0 ml) containing AICA. HC1 (126 ~t mole), cyanamide (1.14 m mole), dTMP (32.8 bt mole) and 3HdTTP (36.2// mole, specific activity 4.2 #Ci//lmole). This solution was neutralized to pH 7.0 with 1.0M NHaOH and as a result NH4CI (126/l mole) was formed from the AICA HC1. The reaction was evaporated at room temperature under nitrogen to dryness and subsequently heated at 60°C for 18 h. Preliminary experiments were performed on a 1.0 ml aliquot of the above solution. For the control reaction, AICA and cyanamide were omitted from the reaction mixture. On completion of the reaction, the products were dissolved in 1.0 ml water and an aliquot of this solution chromatographed on a PEI plate using solvent system I and on Whatman 3-MM paper using solvent systems II and III. Deoxythymidine 5'-phosphate, deoxythymidine and dideoxythymidine diphosphate (pdT)2 were used as markers. The plate or paper was subsequently strip counted to determine the position of the radioactive compounds. A preliminary analysis of the reaction products was obtained by enzymatic degradation procedures as outlined in section 2.3. The bulk of the crude reaction product was subjected to a procedure which degrades pyrophosphate bonds but leaves phosphodiester and phosphomonoester bonds of oligonucleotides intact (Moon and Khorana, 1966). The exact procedure is given in Sherwood and Or6 (1977). Chromatography was again performed on a PEI plate and solvent system I or on Whatman 3-MM paper using solvent systems II or III. Enzymatic analysis of the reaction products remaining after the pyrophosphate degradation procedure was also undertaken. Finally the reaction products were separated into their constituent components using a DEAE cellulose (HCO~form) column using a triethylammonium bicarbonate gradient. This buffer was removed from the various peaks by repeated evaporation of the nucleotide material from water and standard solutions of the peaks were finally prepared containing 5.2 gmole nucleoside units per ml.

3. Results

3.1 Preliminary Experiments In a preliminary experiment, results not shown, we found that a series of oligomeric products were produced as a result of drying and subsequently heating at 60°C for 18 h an aqueous solution of dTTP, dTMP, AICA, cyanamide and NHaCI. The products from this reaction were subjected to the pyrophosphate degradation procedure and subsequently treated with alkaline phosphatase. Chromatography on a PEI cellulose plate using solvent system I indicated that 20% thymidine, based on radioactive measurements only, was obtained, and a series of oligomeric products were evident.

C y a n a m i d e Mediated Syntheses II

197

The results from a control experiment, in which the AICA and cyanamide were omitted from the reaction is shown in Table 1. This table clearly indicates that polyphosphates, unlike dTppT, (Moon and Khorana, 1966) are poorly degraded by the pyrophosphate 1. Analysis o f Products obtained f r o m heating 3HdTTP with dTMP

Table

Reaction

%dTTP

1

91

2 3 4 5

63 53 48 0

%dTDP

%dTMP

3

4

30 30 37 0

8 9 13 0

%dT 0

0 o 0 100

(1) Untreated sample of 3HdTTP. (2) 3HdTTP after pyrophosphate degradation. (3) After a solution o f ~HdTI'P and dTMP was dried and heated at 60 ° C for 18 ja. See section 2.4 for details. (4) The products from reaction 3 after pyrophosphate degradation. (5) The products f r o m reaction 4 after t r e a t m e n t with alkaline phosphatase. The products f r o m these experiments were chromatographed on a PEI cellulose plate using solvent s y s t e m I. They were identified by c o c h r o m a t o g r a p h y with authentic standards. The percentage yields are based on radioactivity m e a s u r e m e n t s only

A

f

f o

D

I

o

2

Jt It

i ~ j,

~

'

,,

i

J

, i

,

xI

~

i

i

, ,

1 i

~

pp

jt j

,

:.

, L

' t

J

i

t

L

d a

8

12

16

cm

Fig. 1. Strip c o u n t s o f the radioactivity of thin layer chromatograms on a PEI cellulose plate, using solvent system I, of the dTTP, dTMP, AICA, CNNH 2 reaction. Each reaction consisted o f dTTP (1.2 ~mole), dTMP (1.2 #mole) AICA.HC1 (4.2 ~mole) and cyanamide in 0.25 ml water. The Final pH o f t h e solution was 4 0. To the reaction (. - ) was added aHdTTP (0.06 # m o l e specific • . . " . 14 ' .. activity 21 mCffmmole) and to the reactaon ( . . . . . ) was added CdTMP (0.01/~mole, specific activity 48.8 m C i / m m o l e ) . The reactions were dried and heated to 90 ° for 18 h. The counts were corrected for background and normalized to a total o f 25,000 counts per rain. The position on the plate of authentic standards o f d e o x y t h y m i d i n e (dT), d e o x y t h y m i d i n e 5 ' - m o n o p h o s p h a t e (pdT), the linear dinucleotide (pT)2 and d e o x y t h y m i d i n e 5'-triphosphate (pppdT) is also shown

198

E.Sherwood et al.

degradation procedure. No apparent condensation occurred when a HdTTP was heated with dTMP in the absence of any condensing agents. If AICA was omitted from the reaction, polymeric products were obtained, but in lower yield (34%). Deoxythymidine 5Ltriphosphate also polymerized when dTMP was omitted from the complete system. The polymeric products from both these reactions have not as yet been investigated.

3.2 Double Label Experiment In a second preliminary experiment, two identical aqueous solutions (pH 4.0) (see Fig. 1 for details of reaction), containing dTTP, dTMP, cyanamide and AICA. HC1 were prepared. To the one solution 3 HdTTP was added, and to the other 14CdTMP. Both solutions were dried and heated to 90°C for 18 h. Figure I shows a strip count of the radioactivity present on a PEI plate after chromatography in solvent system I of the products from the two reaction. The figure clearly illustrates that both labels were incorporated into the polymeric products, but to different extents. Though this experiment was conducted at lower pH and higher temperature than the other reactions described in this paper, it has been included to illustrate that the mechanism of this prebiotic reaction could involve condensation of dTTP on a dTMP or dTppT primer.

,615 176A, ill

x

,'!

30

1

I

~ i

I

'

:

"'°

i

i

,'

7o

2b

i;

o.

'

g

,

g I

20

I 0 cm

30

Fig. 2. Strip counts of the radioactivity of thin layer chromatograms on a PEI cellulose plate, using solvent system I, for the 3HdTTP, dTMP, AICA, CNNH2 reaction before (. - .) after (. . . . . )pyrophosphate degradation. The counts were corrected for background and normalized to a total of 160,000 counts per minute. The numbers 1-7 indicate the positions of the fractions obtained from the DEAE cellulose column (Fig. 4) when chromatographed on the same PEI plate. The position on the plate of authentic standards of deoxythymidine (dT), deoxythymidine 5'phosphate (pdT), the linear dinudeotide (pdT)2 and deoxythymidine 5'-triphosphate (pppdT) is also shown

Cyanamide Mediated Syntheses II

199

20

16

7

o12 3a

,j

8

its2° 5

10

15

1 20

25

30

35

40

in

Fig. 3. Strip counts of the radioactivity of paper chromatograms using solvent system II o f the 3HdTTP, dTMP, AICA, CNNH 2 reaction before (- - .) and after ( . . . . . )pyrophosphate degradation. The counts were corrected for background and normalized to a total of 200,000 counts per rain. The numbers 1 - 3 a indicate the positions o f some o f the fractions obtained from the DEAE cellulose column (Fig. 4) when chromatographed on the same paper. The position on the paper o f authentic standards o f deoxythymidine 5'phosphate (pdT) and the linear dinucleotide (pdT)2 is also shown

Table 2. Analysis o f the Products obtained by heating 3HdTTP with dTMP, AICA and CNNH2

Reaction

%dT

%dTMP

%Products

Apparent %polymer degraded

1 +SV +AP +SP +AP +SP

5.6 a 8.0 a 16.0 8.7 18.0

* * 6.8 30.0

92.0 23.0 84.0 84.5 52.0

-71.0 40.0 10.0 32.0

2 +SV +AP +SP +AP +SP

5.6 4.0 13.0 13.0 13.0

* 72.0 * * *

94.4 24.0 87.0 87.0 87.0

-72.0 24.0 * *

a

69.0

Elution of this material with pH 2 HCI yielded the same OD units

The 3HdTTP, dTMP, AICA, CNNH 2 reaction before (1) and after (2) pyrophosphate degradation. See section 2.4 for details. (SV), snake venom phosphodiesterase digest; (AP), alkaline phosphatase digest; (SP), spleen phosphodiesterase digest; (AP + SP), alkaline phosphatase digest followed, after neutralization with HCI and evaporation to dryness, by digestion with spleen phosphodiesterase. The results for the apparent percentage degradation of the polymeric products were derived by comparing the strip counts from chromatograms, chromatographed in solvent system II or II1, before and after enzymatic digestion. (*) Indicates that due to the complexity of the reaction the yield of this product or products could not be determined

200

E.Sherwood et al.

3.3 Enzymatic Degradation of Polymeric Products before and after the Pyrophosphate Degradation Procedure Encouraged by the results of these preliminary experiments, a large scale reaction (see 2.4 for details) was investigated. Chromatography of the products from the crude reaction mixture, before and after the pyrophosphate degradation procedure, on PE! cellulose plate using solvent system I (Fig. 2) or on Whatman No. 3 paper using solvent system II (Fig. 3) or solvent system llI (not shown) indicated that extensive polymerization had occurred. The results of preliminary analysis of the crude reaction, before and after the pyrophosphate degradation procedure, with various enzymes is shown in Table 2. It should be remembered in interpreting the results from this table, that only the dTTP in the reaction was labeled. Though contamination of nonradioactive components in the reaction with tritium was possible, subsequent analysis showed that the AICA recovered from the reaction had a specific activity of 0.013 ~tCi/bt mole. Since the specific activity of the 3 HdTTP in the initial reaction was 4.1/aCi//a mole, loss of label was not appreciable. Therefore, in general, only the fate of the 3 HdTTP was followed by these degradative procedures.

3.4 Analysis o)'Individual Fractions Obtained from DEAE Cellulose Chromatography It is evident that data derived only from enzymatic digests on bulk polymeric material offers only limited information, which is open to misinterpretation. We, therefore,

1.4

1.0 2a

E

3a

,o

0.8

4 6

0.4

2c

I

2O

I

60 Tube No

I

10LO

I

14hO

Fig. 4. The elution profile obtained from the 3 HdTTP, dTMP, AICA, CNNH 2 reaction, after pyrophosphate degradation, on a DEAE cellulose column using a linear t r i e t h y l a m m o n i u m bicarbonate gradient. For identification of t h e various fractions see Table 7

Cyanamide Mediated Syntheses II

201

p r o c e e d e d t o separate t h e p o l y m e r i c p r o d u c t s after p y r o p h o s p h a t e d e g r a d a t i o n o n a D E A E cellulose c o l u m n , b i c a r b o n a t e f o r m (Fig. 4), (see e x p e r i m e n t a l section f o r details). E a c h o f t h e m a j o r f r a c t i o n s o b t a i n e d f r o m this c o l u m n were c h r o m a t o g r a p h e d in solvent s y s t e m s I, II and III. In all cases e x c e p t 2a a n d 2b, greater t h a n 90% o f t h e r a d i o a c t i v i t y c o i n c i d e d w i t h a single U V a b s o r b i n g s p o t . Table 3 gives t h e R f values o f t h e m a j o r f r a c t i o n s in solvent s y s t e m s I, II a n d III. The radioactive, U V a b s o r b i n g s p o t s f r o m t h e s e c h r o m a t o g r a m s were e l u t e d w i t h a k n o w n v o l u m e o f p H 2 HC1 and t h e r a d i o a c t i v i t y o f t h e e l u t e d material p e r OD u n i t per ml at 270 n m d e t e r m i n e d . Table 4 indicates t h a t with t h e e x c e p t i o n o f

Table 3. Rf and Em Values of the Various Fractions obtained from the DEAE Cellulose Column (Figure 4) Solvent System a II (Rf) III (Rf)

Fraction No.

I (Rf)

1

0.95

2a

0.80

0.63 0.50 (NR) 0.40

2b 3a 4 5 6

0.81 0.48 0.41 0.28 0.18 -

0.17 0.30 0.21 0.11 0.07 0.03

7 dT dTMP dTpT AICA

0.93 0.81 -0.90

0.02 0.67 0.17 0.46 0.50

IV (E m) b

0.58 0.48 0.38 0.42 0.23 0.17 0.15 0.12 0.09 0.17 (NR) 0.07 0.67 0.42 0.48 0.58

0.00 0.43

a See section 2.2 for description of chromatography and solvent systems b Mobdlty - - relative • to deoxythymidine 5 -phosphate (NR) Non radioactive

Table 4. Observed CPM/OD unit at 270 nm/ml of the Major Fractions from the DEAE Cellulose Column (Figure 4)

Fraction No.

CPM/OD unit at 270 nm/ml Found

1 2a 2b 3a 4 5 6 7

1900 (Rf solvent system II, 0.63 48000 57000 93000 128000 165000 201000 (Rf solvent system III, 0.09) 235000

Calc. a

134500 179300 201800 215200 224200 230500

The major fractions from the DEAE cellulose column were further purified by paper chromatography using solvent systems II or IlL The radioactive, UV absorbing spots were eluted from the paper with a known volume of pH 2 HC1 and the OD at 270 nm and the radioactivity of the eluted material determined a

These values were calculated for oligomers exclusively of the type dT(p3HT)n where n = 1--6. ( 1 0 D unit at 270 nm/ml 3HTTP gave 269000 CPM.)

202

E.Sherwood et al.

fraction 1, the increase in radioactivity of the remaining fractions formed, in general a homologous series, with an average increase of 37,000 CPM per fraction number. The UV absorption spectra of these purified fractions were a composite of a quaternary pyridinium cation and a thymidine chromophore (identification discussed in Sherwood and Or6, 1977). When the fractions were degraded with snake venom phosphodiesterase and the enzymatic products analyzed by paper chromatography it was found that the liberated dTMP had the normal UV absorption of a thymidine chromoclose to that phore, while the nucleotide material which chromatographed with an of deoxythymidine had the pyridinium group attached to it. This indicated that the pyridinium group was attached to the 5'-OH end of these oligomers. The introduction of this group occurrred during the pyrophosphate degradation procedure in which pyridine was used as a solvent. The presence of a pyridinium group on these oligomers is of little consequence in relation to the primary reaction, but it did make subsequent enzymatic analysis of the type of bonding in the oligomers more difficult. Table 5 gives the results from digestion of fractions 2a through 7 with snake venom phosphodiesterase, followed by estimation where possible, of the ratio, dTMP/ "pyr"dT, by optical density measurements, after elution (pH 2 HC1) of the spots which chromatographed with dTMP and "pyr" dT in solvent systems II or III. The results from fractions 2a, 3a and 5 are consistent for oligomers of the type " p y r " d T (pT) n where n = 1, 2 or 4 respectively. The ratio of CPM of 3 HdTMP to "pyr ''3 HdT for the various fractions after digestion with snake venom phosphodiesterase are also recorded in this table. The values obtained for fractions 2a, 3a, 5 and 7 form a homologous series, the 5'-terminal deoxythymidine residue derived mainly from the unlabeled dTMP and the remaining residues mainly from 3 HdTTP. If unlabeled dTMP was not incorporated into these oligomers, a ratio of I instead of 2.3 would be expected for fraction 2a and a ratio of 2 instead of 4.3 would be expected for fraction 3a, etc. The 3 HdTMP/,,pyr,,3 HdT ratios for fractions 4 and 6 do not conform with this series. The high values may be explained on the assumption that these fractions contained, in addition to oligomers of the type "pyr"dT(pT) n, where n = 3 or 5, oligomers with

Rf

Table 5. Analysis of the Major Fractions from the DEAE Cellulose Column (Figure 4) with Snake Venom Phosphodiesterase

Fraction

dTMP OD of spot/ml 270 nm

2a dTpTa 3a dT(pT)2 4 dT(pT) 3 5 dT/pT) 4 6 dT(pT) 5 7 dT(pT)6

0.46 1.26 1.76 1.12 # #

"pyr"dT OD of spot/nil ratio 270 nm found 0.44 0.61 0.28 0.27 # #

1.05 2.06 6.34 4.14 ---

dTMP/dT theor, 1 2 3 4 ---

3HdTMP/"pyr"3HdT ratio 2.3 4.3 38 9.5 40 13.7

The products from the enzyme digests were chromatographed on paper using solvent system II. The UV absorbing, radioactive spots which cochromatographed with dTMP and dT were eluted from the paper with a known volume of pH 2 HC1 and the OD at 270 nm and the radioactivity of the eluted material determined. a See Table 8 for details. ( # ) Insufficient material for analysis

Cyanamide Mediated Syntheses 11

203

5'5'-pyrophosphate bonds. The pyrophosphate degradation procedure does not degrade pyrophosphates completely, so contamination of some of the fractions with oligomers containing pyrophosphate bonds is to be anticipated. Partial degradation of fractions 5, 6 and 7 using snake venom phosphodiesterase yielded in each case a series of oligomers corresponding to the number of nucleotide units in the initial oligomer analyzed. These results confirmed that these fractions contained oligomers of the type "pyr"dT(pT)n, where n = 4, 5 or 6. The results obtained from the various fractions after digestion with alkaline phosphatase snake venom phosphodiesterase and spleen phosphodiesterase are given in Table 6. The fact that the majority of these oligomers were readily degraded by snake venom phosphodiesterase and largely untouched by alkaline phosphatase or spleen phosphodiesterase is consistent for oligomers containing a blocking pyridinium group in the 5'-OH position. A detailed analysis of the nucleotide material present in the various fractions is given below. We have summarized the results of this analysis in Table 7 t@ether with the yield of nucleotide material present in the prebiotic reaction after pyrophosphate degradation as estimated by the OD value at 267 nm of the various fractions after elution from the DEAE cellulose column.

3.5 Detailed Analysis o f the Individual Fractions from the DEAE Cellulose Column Fraction 1. Fraction 1 yielded two UV absorbing spots on paper chromatography in solvent system II (Table 3). The upper spot, which contained most of the radioactivity present in fraction 1, chromatographed slightly below deoxythymidine in solvent system II and with AICA in solvent system III. The lower spot co-chromatographed with AICA in solvent systems II and III and gave the characteristic pink color of AICA on air drying the paper overnight. The UV absorption spectrum of the radioactive material in pH 2 HC1 had the general appearance of a composite spectrum of deoxythymidine and a N-methylpyridinium cation. We discuss attempts to clarify the composition of this compound in the preceding paper (Sherwood and Or6, 1977). The results obtained indicated that this compound was probably 5'-C-pyridinium deoxythymidine or a closely related deoxythymidine derivative.

Table 6. Enzymatic Degradation of the Major Fractions from the DEAE Cellulose Column (Figure 4) Fraction

Snake venom % degradation

Spleen % degradation

Alk. phos. % degradation

2a

87

39

3a

100

9

0 0

4 5 6 7

96 100 84 88

4 3 2 4

4 3 1 2

The percentage yields are based on radioactivity measurements only

204

E.Sherwood et al.

Fraction 2a. An analysis of the nucleotide material present in fraction 2a, based on the results of paper chromatography, electrophoresis and enzymatic digestion of the individual components present in this fraction, is given in Table 8. We have concluded that the component of this fraction (24%) which had an Rf of 0.48 in solvent system III and an E m of 0.00 in solvent system IV was "pyr" dTpT. Its UV spectrum, electrophoretic mobility, resistance to digestion by spleen phosphodiesterase and digestion by snake venom phosphodiesterase to yield dTMP and "pyr" dT (Sherwood and Orr, 1977) only, is in accordance with this conclusion. The remaining nucleotide material ( R f solvent system III, 0.38 and Em, solvent system IV, 0.43) consisted of AICA-pdT (37%) and dTpT (39%). This conclusion is based on the complete digestion of this material with snake venom phosphodiesterase to yield AICA, dTMP and deoxythymidine and partial degradation (39%) by spleen phosphodiesterase. The OD units at 270 nm/ml for dTMP, derived from AICA-pdT, was calculated from the OD/ml for AICA obtained from the snake venom phosphodiesterase digest of the total fraction. Hence a value of 0.46 (Table 6) was calculated for the OD units at 270 n m / m l for dTMP obtained from "pyr" dTpT and dTpT after digestion with snake venom phosphodiesterase. A value of 1.05 for the ratio dTMP/dT for the total dinucleotide material in the fraction was derived. Similarly, by excluding the contribution of AICA-pdT

Table 7. The Products obtained from the DEAE Cellulose Column (Figure 4) Total OD Polymers Fraction Tube No. at 267 nm % Identification a 1

3--6

168

9.1 b

2a

11-16

48

18.1

2b

22-27

34

13.1

2c 2d

33-36 42-46

8.5 9.4

3.3 3.6

3a

51-55

26.7

10.4

3b

62-65

6.4

2.5

4

77-82

30.8

12.0

5

100-109

28.5

11.0

6

121--128

20.2

7.8

7 8

134-141 147--152

13.7 7.7

5.4 3.0

86% AICA 14% "pyr" dTc 37% AICA-dpT 39% dTpT 24% 5'-C-"pyr"dTpT c 30% dpT 70% cyclo(dpT) 2 not identified not identified 9% dT(pT)2 91% 5'-C-"pyr"dT(pT)2 c not identified 5'-C-"pyr"dT(pT)2 Analyzed + side product d 5'-C-"pyr"dT(pT)4 c 5'-C-"pyr"dT(p.T)s + side product tl 5'-C-"pyr"dT(pT) 6 not identified

a Thesepercentage are based on radioactivity measurements, with the exception of fraction 1 where OD measurements were used b This percentage is for "pyr"dT only e The assignment of a pyridiniumgroup at the end of these oligomers is tentative, see text for details d For discussion on nature of the side products see text for details

Cyanamide Mediated Syntheses II

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