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Selectivity in aqueous peptide synthesis by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
Biochimica et Biophysica Acta, 295 (1973) 385-395 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA36318 SELECTIVITY IN AQUEOUS PEPTIDE SYNTHESIS BY I_ETHYL_3_(3-DIMETHYLAMINOPROPYL) CARBODIIMIDE A MODEL FOR PRIMORDIAL PROCESSES
MARIAN E. ADDY*, GARY STE1NMAN** ANI~ M. F. MALLETTE
Department of Biochemistry, The Pennsylvania State University University Park, Pa. 168o2
(U.S.A.) (Received June 23rd, 1972) (Revised manuscript received October 24th, 1972)
SUMMARY
Commercial vasopressin was purified, the terminal glycinamide removed by tryptic hydrolysis and the resulting des-glycinamide vasopressin was isolated and employed as a model in studying selectivity in peptide bond formation. Water soluble i-ethyl-3-(3-dimethylaminopropyl) carbodiimide was used to condense various individual aliphatic amino acids to the C-terminal end of des-glycinamide vasopressin forming analogs of 81ysine-vasopressin. Thin layer chromatography on Silica Gel G, paper chromatography and paper electrophoresis were combined to isolate peptide products. They were characterized by end-group analysis and tryptic hydrolysis. There was a general increase in yield with size of the aliphatic side chain. Although glycinamide occupies the carboxyl terminal position of lysine vasopressin, this amino acid derivative did not condense with des-glycinamide vasopressin. Nor did either of two other amides. Moreover, glycine condensed to a lesser extent than did the other amino acids used. In addition, a-N-acetyllysine was used as a second model acceptor for amino acids, this time representing the penultimate amino acid residue of vasopressin. It showed a preference for glycine over some other amino acids. The limited selectivity observed in the various condensations is attributed to the microenvironment at the reaction center, since the amino acids studied were chemically similar in their side chains and included glycine, sarcosine, alanine, aaminobutyric acid, valine, norvaline, leucine, isoleucine, and norleucine. It is suggested that prebiological synthesis of peptides by means of such condensation reactions led to somewhat higher concentrations of preferred ploducts, some of which were active in developing biological systems. * Present address: Department of Biology, Federal City College, Washington, D.C. 20001, U.S.A. ** Present address: Ames-Yissum Ltd., Shattner Center Building 3, Jerusalem, Israel.
386
M.E. ADDY et al.
INTRODUCTION
Chemical evolution concerns geochemical synthesis of molecules essential to life and focuses attention especially on nucleic acids, proteins and their precursors. Prebiological synthesis of proteins involves formation of peptide bonds in the absence oI the system presently used, based on enzymes, ribosomal assembly, and sequence information in messenger RNA. The formation of a peptide bond is a dehydrating condensation with a free energy requirement of about 4 kcal/mole (ref. i). This energy barrier is overcome biologically by utilization of energy transfer intermediates. Under primordial conditions, energy might have been obtained by coupling peptide bond synthesis with an exergonic hydrolysis which provided the necessary free energy. Carbodiimides are highly tea ctive condensing agents ~. Yet they could have been present in primordial times as isomers of protonated cyanamide anion 3. Selective incorporation of amino acids in such a system could have led to peptides and proteins with primitive biological functions. Evidence supporting this possibility has been obtained 4 from dipeptide syntheses under simulated primitive earth conditions. These dicyanamide generated dipeptides had compositional frequencies corresponding 4 to the occurrence of such dipeptides in present day proteins. It has been suggested 5 that selectivity in chemical synthesis of peptides is due to differences in side chains of the amino acids and led to some orderliness during prebiological peptide synthesis. The present investigation attempts to evaluate some of the factors which might have been responsible for this postulated orderliness. Because of its availability and convenient chemical features, the peptide hormone ~lysinevasopressin was taken as a starting point. The concept underlying this portion of the work is shown diagramtically in Fig. I. In this approach the terminal glycinamide residue was removed by tryptic hydrolysis yielding an octapeptide called desglycinamide vasopressin. Condensation of this peptide with simple aliphatic amino acids was studied as an indicator of preferential amino acid incorporation. Some selectivity in the condensation would be expected because of the proposaP that any peptide at a given state of growth may have enough chemical information to favor conditionally tile addition of particular amino acids. Moreover, the carboxylterminal amino acid of the peptide by its special position would be expected to influence the condensation by selectivity with respect to the next amino acid added. Thus, there should be two factors cooperating, the growing peptide as a whole and the carboxyl-terminal amino acid. In the present study, an attempt is made to distinguish between these two factors by employing both des-glycinamide vasopressin and a lysine derivative representing the penultimate amino acid in vasopressin, a-N-Acyllysine was used as the lysine derivative resembling as closely as possible the lysine residue in des-glycinamide vasopressin. Preliminary experiments revealed that glycinamide did not condense with desglycinamide vasopressin in the reaction system used here, nor did it condense with aN-acetylysine (Addy, M. E., Steinman, G. and Mallette, M. F., unpublished results). Therefore, tree amino acids were used in place of their amides in this study. While such a choice of reactants does not take account of present day genetic coding for glutamine and asparagine, it can be justified by absence of DNA coding for any other amides or for glycosyl groups in glycoproteins. Presumably, amino acids are incorpo-
MODEL FOR PRIMORDIAL PEPTIDE SYNTHESI~
387
rated first in free form and derivatized later. Thus, amidation of glycine to form vasopressin after first condensing the amino acid may be analogous to acetylation of the e-amino group of lysine observed 7 for certain histones or the removal of formyl groups from terminal N-formylmethione followed by hydrolytic cleavage of one or two residues from the precursors of many bacterial proteins s. MATERIALS AND METHODS
Free amino acids, I-dimethylaminonaphthalene-5-sulfonyl choride (dansyl chloride) and qysine-vasopressin were obtained from Sigma Chemical Co. a - N Acetyllysine was purchased from Calbiochem, a-N-tosyllysine from International Chemical and Nuclear Corp., and I-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride from Cyclo Chemical Co. Trypsin twice crystallized was obtained from Mann Research Laboratories. Sephadex G-I5, particle size 4o-12o, from Pharmacia Fine Chemicals, and [1-1*C]leucine, [i-14C]glycine and [I-l~C]valine were obtained from International Chemical and Nuclear Corporation and Tracerlab. Dipeptides of a-N-acyllysine and aliphatic amino acids were synthesized as follows. Each reaction mixture consisted of o.I ml of 0.06 M a-N-acetyllysine or a - N tosyllysine in o.I M HC1, o.I ml of 0.06 M amino acid in water from the list following and o. I ml of o. I M I-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The amino acids used individually were glycine, alanine, sarcosine, valine and norvaline, leucine, isoleucine, and norleucine. The carbodiimide was added last, and the reaction mixture held overnight at room temperature. In other experiments, 14C-labeled amino acids were mixed with their 12C forms before combination with other reactants. The radioactive amino acids then served as quantitative and qualitative control measures of reaction of unlabeled products. Des-glycinamide vasopressin was prepared as follows. Commercial vasopressin was relatively impure with an activity of about IOO I.U./mg, compared to about 5o0 I.U./mg for pure material. Vasopressin in water was added to trypsin and buffer solutions in a volume ratio of 3 :I :26. The buffer was 0.046 M Tris-HC1 (pH 8.1) and contained o.oi15 M CaCI~. Trypsin was in o.ooi M HC1 and was in a weight ratio to vasopressin of 1:50. Hydrolysis was continued for 24 h at 30 °C and stopped by addition of a small volume of I M HC1. The hydrolysis mixture was lyophylized and a small sample of the residue taken up in o.I M NaHCOs. This solution was used to identify products in the hydrolysate using the dansylation procedure of Bailey 9 for determination of end-groups. Dansyl derivatives were separated by thin-layer chromatography on silica gel G using the upper layer formed after equilibration of diisopropyl ether-2-butanone-acetic acidwater (8:2:5:5, by vol.). The rest of the lyophylized material was taken up in 50% acetic acid. Des-glycinamide vasopressin was isolated from this solution and purified by gel filtration according to the procedure of Manning et al. ~o developed for purification of oxytocin. Tyrosine-containing peptides from the column were located by a modified Lowry method n. Fractions containing such peptides in the major peak were pooled, diluted with 2 vol. of water, lyophylized to dryness and the residue taken up in 0.5 ml of 0.2 M acetic acid. This solution was applied to the same Sephadex column previously re-equilibrated with 500 ml of 0.2 M acetic acid. Fractions were again checked for peptide con-
3 H8
M . E . ADDY ,.'{ ct[.
tent by the modified l.owrv method after addition of an equal volume of (t.2 M NaOH. "File only majot component, believed to be tile desired product, was diluted with e vol. of water and lyophylized. File white fluffy peptide was analyzed for its amino acid content alter hydrolyzing it as follows. Approx. o.o5 mg was dissolved in o.5 ml of () M H i l , sealed in glass under Ne, and heated for ~2 h at IIO :('. The hvdrolvsate was diluted with about 5 vol. of water, evap~rated to dryness iH ~'acem, and taken up in a small volume of water, This solution is chromatographed on Whatman N~. i sheets using /er/dmtanol 2-butanone formic a(id \rater (4o:3o:i5:i5, by vo].), i-butanol acetic acid- water (8o :2o: 2o, by vol.), and phenol-water (8o :2o, w/v). Papers were sprayed with o,42;, ninhvdrin in absolute ethanol and colors developed for about 2 rain at H o "C. The amino acids of vasopressin were included as standards in their free forms. In addition, two dimensional paper chromatography, was en~ployed using the phenol-water and >butanol-acetic acid water systems. Analogs of vasopressin were syntbesized from des-glycinamide vasopressin by dissolving the latter in water to approx. ().oI M and adding o.oe5 ml to o.oI5 m! of o.o5 M solution of an amino acid. To this mixture was added ,.~.oi ml of a solution of that same amino acid labeled with 1~('. Then o.o2 5 ml of o.t M I-ethyl-3-(3-dimethylaminopropyl) earbodiimide was added and the reaction mixture held at room temperature for 4 11. I,ow voltage paper electrophoresis on \,Vhatman No, z paper with borate buffer *e at pH 9.2 was used to fractionate condensation mixtures containing radioactive components. After applsdng 3oo V for I2 h, paper strips were dried, and radioactive materials were located by exposure to X-ray fihu for I2 7 ~ h. Reaction mixtures were further fraetionated by paper chromatography as described above and bv thin layer chromatography on o.2 5 mm of Silica Gel (; on glass plates. Reaction mixtures and accompanying standards, were developed on the thin-laver plates at room temperature for about 4 I1 with I-butanol acetic acid water ({%:_m:e~;, by x~d.) or /crt-butanol--2-butanone water NH4()tl (4o:3o:2o:~o, by vol.). After air drying, separated components were detected bv ninhvdrin spray. Both chromatographic sheets and thin-layer plates containing radioactive materials were examined by exposure to X-ray film for I2 7 2 h after covering the plates with Saran \Vrap. After larger scale syntheses and separations, materials thought to be peptides were eluted and c(mcentrated to small volumes for analysis. Dipeptides isolated from condensation systems by thiu-layer chrmnatography and paper electrophoresis were studied by dansylation' and acid hydrolysis. Products of hydrolysis were separated by thin-layer c}~romatograpby and identified by cochron> atography with standard amino acids. Dansvl derivatives were identified by cochromatography with dansyl derivatives prepared from individual components used in the synthesis. In agreement with another study (Addy, M. E., Steinman, G. and Mallette, M. F., unpublisiled results) e-N-aminoacyllysine dipeptides were not (ibserved. Rather, the condensations involved the ('-terminus of (,-N-acetyllysine. Analogs of vasopressin were isolated by paper electrophoresis using X-ray film to locate them. Each was hydrolyzed by trypsin and the liberated amino acid identified by thin-layer chromatography of the hvdrolvsate and eochromatography with the corresponding reference amino acid. Vasopressin analogues and des-glyeinamide vasopressin were also detected b \ an acidic palladium chloride spray specific ~a fro_ oxidizable sulfur. Ninhydrin spray was employed for detection of components in the absence of radioactivit\.
MODEL FOR PI~IMORDIAL PEPTIDE SYNTHESIS
389
Peptides not containing radioactive amino acids were quantitated by densitometry of ninhydrin-positive components on thin-layer plates with a Photovolt nmltiplier photometer Model 52o-A with stage attachment for the plates and a Varicord Model 423 recorder. Peak area was used as a measure of peptide weight. This method measures the dipeptide as a function of the intensity and size of colored spots produced on thin-layer plates by ninhydrin spray. If the wavelengths of maximum absorption by the colored product differ with the amino acid, somewhat different color intensities would be expected at equal concentrations and any one wavelength. In this instrument the distribution of wavelengths is constant. Therefore, the data were normalized by dividing areas under peaks due to dipeptides by corresponding reference areas for the amino acid present in an individual peptide. This procedure assumes that condensation does not shift the wavelength of maximum absorption. Thus, data reported after ninhydrin treatment are not actually yields because of this procedure. Moreover, there is a contribution to color by the a-N-acyllysyl group. Since this moiety was present i n all peptides assayed in this way, the results are comparable for related condensations. Radioactive peptides on paper were determined by cutting out zones located with X-ray films and counting in Packard liquid scintillator with a Beckman Model LS-2ooB scintillometer. Data are also reported as yields relative to glycine (a-aminobutyric acid in one table) to make more manifest the trends in incorporation of amino acids. RESULTS AND DISCUSSION
a-N-Acetyllysine was condensed in groups of six different reaction mixtures with individual amino acids from the following series: a-aminobutyric acid, valine, norvaline, leucine, isoleucine and norleucine. Glycine, alanine and sarcosine form dipeptides which did not separate from the corresponding flee amino acids in the chromatographic systems used. Therefore, these three amino acids also were condensed with a-N-tosyllysine. Presence of this acyl group permitted chromatographic separation of products and residual reactants and made yield comparisons possible. Furthermore, for evaluation of any effects of the two different protecting groups on yields of dipeptides, radioactive glycine, valine and leucine were condensed with a-Nacetyllysine. The products were successfully isolated by electrophoresis and assayed by determining the radioactivity. Results from these three different sets of syntheses are presented in Tables I, II and III. The data of Table I indicate similar Yields for a-N-tosyllysylglycine, a-Ntosyllysylsarcosine and a-N-tosyllysylalanine. Therefore, condensation of a-N-acetyllysine with glycine should yield product to about tile same extent as for a-N-acetyllysylleucine in accord with the absolute yield data of Table II. On the other hand, comparisons of yields for the dipeptides of glycine in Tables I and II and of leucine in Tables II and III show that the acetyl and tosyl protective groups did make a difference in yield. Data on yields relative to giycine may be compared from the first three tables for the nine amino acids employed. The maximum difference (between norvaline and norleucine) in relative yield is about 65 %. General trends in selectivity during synthesis of a-N-aeetyllysyl dipeptides of of aliphatic amino acids are summarized as follows.
39 °
M.E. ADDY el al.
"FABLE I Y I E L D S OF ( 2 - ~ - T O S Y L L Y S I N E D I P E P T I D E S
R e a c t i o n s as in T ab le 11 [ e x c e p t t h a t a - N - t o s y l l y s i n e r e p l a c e d (z-N-acetyllysine. D a t a are m e a n s of s e v e n r e p l i c a t e e x p e r i m e n t s (eight for sarcosine).
(;lycine
.~;arcosine Alanine
18, l i .5 o I.OO
17. 7 1.14 o.98
R e l a t i v e y ield Standard deviation Yield r e l a t i v e to g l y c i n e
t8,i 1.75 i.oo
T A B L E I1 YIELDS OF RADIOACTIVE Cl-N-ACETYLLYSYLIIlPEPTIDES
R e a c t i o n s as in T a b l e I I I e x c e p t t h a t t r a c e r a m o u n t s of [I-14C]-labeled a m i n o a c i ds were a d d e d a l o n g w i t h t h e c o r r e s p o n d i n g u n l a b e l e d forms. R a d i o a c t i v i t y w a s d e t e r m i n e d in 5 ° p l of r e a c t i o n m i x t u r e before a n d a f t e r f r a c t i o n a t i o n to isolate t h e d i p e p t i d e s . Y i e l d s w e re c a l c u l a t e d as (14C in dipeptide/14C in r e a c t i o n m i x t u r e ) × IOO a n d are r e p o r t e d as t h e m e a n s of four r e p l i c a t e experiments.
Mean y i e l d (°/o) Standard deviation Y i e l d r e l a t i v e to g l y c i n e
Glvcine
Valine
Leucine
13. 4 o, 14 t,oo
II. 4 o. 23 0,8 5
14. 4 o. i i 1.o8
TABLE llI P E P T I D E Y I E L D S R E L A T I V E TO G L Y C I N E
E a c h r e a c t i o n m i x t u r e w a s o.o2 M a - N - a c e t y l l y s i n e , o.o33 M HC1, o.o2 M a m i n o a c i d a n d o.o33 M i - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l) c a r b o d i i m i d e . H e l d o v e r n i g h t a t r o o m t e m p e r a t u r e . D a t a are r e l a t i v e p e r c e n t a g e s o b t a i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s a n d a re m e a n s of s e v e n rep l i c a t e e x p e r i m e n t s . Y i e l d s r e l a t i v e to g l y c i n e are c a l c u l a t e d be l ow f r o m v a l u e of T a b l e I I for leucine usin g t h e r a t i o of 1.o8/I.42.
Relative yield Standard deviation Y i e l d r e l a t i v e to a - a m i n o b u t y r i c acid Y i e l d r e l a t i v e to g l y c i n e of T a b l e s I [ and III
a-Aminobutyric acid
Valine
Norvaline
Norleucine
Leucine
Isoleucine
lO.6 o.71
lO.8 o.7o
io.i 1.13
16.6 o.66
15.o o.73
12.o o.5o
i.oo
1.o2
0.95
1.57
1.42
I,I3
0.76
0.78
0.72
1,2o
1.o8
0.86
Except for the three smallest amino acids, glycine, alanine, and sarcosine, yield of dipeptide tended to increase with size of the side chain (Table III). Moreover, relatively high incorporations of glycine, alanine, and sarcosine as inferred by comparisons in Tables I and U with data for leucine suggest a second kind of size effect. Perhaps the kinetic mobility of the smallest amino acids permits more frequent collisions and leads to higher yield. Since all the amino acids used possess inert side chains, the selectivity among them cannot be due to chemical reactivity of the side chains. Rather, it must be due to size and geometry. Comparison of yields obtained from leucinc, isoleucine, and
MODEL FOR PRIMORDIAL PEPTIDE SYNTHESIS
391
norleucine e m p h a s i z e s t h e possibility for a role of side chain g e o m e t r y . The m e c h a n i s m p r o p o s e d 3 for this t y p e of c o n d e n s a t i o n suggests t h a t t h e i n t e r m e d i a t e for t h e last s t e p is t h e s a m e in all syntheses. I t is t h e p r o d u c t of a - N - a c y l l y s i n e a n d condensing agent. Therefore, diffeI ences in yield should be due to a m i n o acids a d d e d C-terminally. A p a r t from a n y differences in i n h e r e n t b a s i c i t y of the amino groups condensing, the y i e l d should d e p e n d also on t h e accessibility of these groups for t h e nucleophilic d i s p l a c e m e n t reaction. A n y crowding a r o u n d the amino g r o u p should lower the yield. I n t h e series norleucine, leucine a n d isoleucine, crowding increases in this order, a n d yield decreases in t h e same order as shown b y T a b l e I I I . The r e l a t i v e yields of d i p e p t i d e s show t h a t some s e l e c t i v i t y is expressed b y a - N acetyllysine as the p e n u l t i m a t e amino acid of vasopressin. I t is modified b y p h y s i c a l factors of t h e a m i n o acids condensing into t h e C-terminal position. This s e l e c t i v i t y is n o t t h e s a m e as t h a t o b s e r v e d 4 in o t h e r similar d i p e p t i d e syntheses. P r e s u m a b l y the charge on lysine is responsible for the difference in b e h a v i o r c o m p a r e d to g l y c y l dipeptides. I n these different b u t r e l a t e d systems, t h e r e was a t r e n d 4 t o w a r d r e d u c e d i n c o r p o r a t i o n into d i p e p t i d e s as a m i n o acids increased in size. TABLE IV YIELDS
OF
PEPTIDES
FORMED
BY
CONDENSING
AMINO
ACIDS
C-TERMINAL
TO DES-GLYCINAMIDE
VASOPRESSIN
The reaction mixture was 0.o 5 M in amino acid, o.oi M in des-glycinamide vasopressin, o.I M in I-ethyl-3-(3-dimethylaminopropyl) carbodiimide and o.i M in HC1. Initial pH was 2.5 and reaction time 4 h. Yields were calculated as in Table II except that the product reported was [I4C]vasopressin analog. There were five replicates of the leucine experiment, four of each of the others.
Mean yield (%) Standard deviation Yield relative to glycine
Glycine
Sarcosine
Leucine
2.54 o. 16 1.oo
1.32 0.o 5 o,51
3.6o o.22 1,42
TABLE V YIELDS OF PEPTIDES VASOPRESSIN
FORMED
BY
CONDENSING
AMINO
ACIDS
C-TERMINAL
TO DES-GLYCINAMIDE
The reaction mixture was that of Table IV except that des-glycinamide vasopressin was o.oo7 M. Yields calculated as in Tables I I I and IV, The glycine and leucine experiments were in three replicates, the others in five.
Mean yield (%) Standard deviation Yield relative to glycine
Glycine
Alanine
Valine
Leucine
Isoleucine
1.o5 0.o9 i,oo
1.29 o.o6 1,23
1.41 o.02 1.34
1.85 o.o 7 1.76
1.72 0.02 1.63
Yields are given in Tables I V a n d V for condensation o f des-glycinamide vasopressin w i t h v a r i o u s flee a m i n o acids. Different p r e p a r a t i o n s of des-glycinamide vasopressin were used for t h e e x p e r i m e n t s o f the two tables. I n general, yields o f the n o n a p e p t i d e s were low c o m p a r e d to yields in d i p e p t i d e syntheses, even t h o u g h the r e l a t i v e a m i n o acid c o n c e n t r a t i o n was higher. These lower yields would be e x p e c t e d because the size of d e s - g l y c i n a m i d e vasopressin should m a k e it react more slowly
302
M.V. AI)I)Y el ~1,
S
S
I
I
Cys-Tyr-Phe- Gin Asn-Cys -Pro Lys-GIyNH2 Lysine-vasopressin I Tryptic hydrolysis S
S
I
I
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys+ GlyNH2 Des-glycinamide vasopressin
RCHCOOH(NH2) NH2 + l-ethyl-3-(3-dimethylaminopropyl)carbodiimide Vasopressin analog Fig. i. Formation of des-glycinarnide vasopressin and its utilization in peptide bond synthesis Experimental details are given in Materials and Methods. Free amino acids were condensed with the carboxy terminal lysine residue. Attempted condensations with amino acid amides were unsuccessful.
with condensing agent than did a-N-acyllysine. Perhaps more influential, yields ol vasopressin analogs may be significantly reduced by a tendency for the condensation to occur N-terminal with des-glycinamide vasopressin, thereby attaching the new residue at the available N-terminal position. Information on the extent of this reaction would be a useful adjunct in interpreting the geometric effects on selectivity in the formation of vasopressin analogs. A general comparison of the yields of Tables IV and V suggests a strong concentration dependence. \Vhile different preparations of des-glycinamide vasopressin were used in these two experiments, this difference should not be an important factor because of the extensive isolation-purification procedure employed. Because of the limited supply, octapeptide concentration was reduced from o.oi to o.oo7 M. This change might lower materially the yield of vasopressin analogue while at the same time increasing yields of such side-products as the decapeptide with amino acid condensed to both ends of des-glycinamide vasopressin. With amino acid and condensing agent concentrations unchanged, by-products should increase rapidly in yield as concentration of des-glycinamide is reduced. Table IV reveals a lower incorporation of sarcosine than glycine. This effect might be due to steric interference by the N-methyl in condensations with the relatively large carboxyl donor, des-glycinamide vasopressin. An incoming group must approach the large acylcarbodiimide intermediate from a limited direction. The Nmethyl group of sarcosine might hinder this necessary approach. Of the amino acids examined in the experiment of Table V, incorporation into analogs of vasopressin increased in the order glycine, alanine, valine, isoleucine, and
MODFL FOR PRIMORDIALPEPTIDE SYNTHESIS
393
TABLE VI PEPTIDE
YIELDS
RELATIVE
TO G L Y C I N E
Yield data obtained according to Materials and Methods are taken from Tables I-V. They are related to glycine by dividing percentage yield by the corresponding value for glycine. Since the experiment of Table III did not contain glycine, data reduction for that table involved equalizing the value for leucine to that for leucine in Table II by dividing by the factor 13.9 calculated as the expected yield for glycine in this series from (15.o) (13.4)/(14.4) in which the factors are respectively yield of leucine dipeptide in Table III and yields of glycine and leucine derivatives in Table lI. C-Terminal Glyeine amino acid
Sarcosine
dlanine
Valine
Leucine
0.85 0.78
1.08 1.08
1.34
1.42 1.76
a-Aminobutyrate
z%Torvaline
Norleucine
Isoleucine
0.76
0.72
1.20
0.86
a-N-A cyllysyl dipeptides
N-Acyl group Tosyl Acetyl
1.oo 1.oo
0.98
1.00
Vasopressin analogues
i.oo t.oo
o.51 1.23
leucine. While glycinamide occupies this position in the natural hormone, of the amino acids with free primary a-amino groups, glycine was incorporated to the smallest extent, Moreover, incorporation increased with size of the R-group. Slightly diminished incorporation of isoleucine relative to that of leucine may be attributed to the slightly less favorable geometry of the R-group and its interference with nucleophilic attack of the amino group. It will be recalled that leucine was more extensively incorporated than isoleucine into a-N-acetyllysyl dipeptides (Table III). Preference for amino acids with larger R-groups is not likely to be due to increased hydrophobic character of larger aliphatic side chains. The reactions visualized ~ in the peptide synthesizing system are not likely to be affected by hydrophobic groups. Furthermore, if there are pK differences for these aliphatic amino acids, they are too small to be significant in these condensations. Moreover, the listed differences ~4 do not correlate with the yield differences of Table V. Table VI compares the selectivity differences discussed above for syntheses of a-N-acylysyl dipeptides with those for the larger vasopressin analogues. Data are normalized with respect to glycine to facilitate the comparison. Note that there is a maximum selectivity factor of about three in this series of aliphatic amino acids. Vasopressin analogues containing leucine were formed at des-glycinamide vasopressin concentrations of o.oi and 0.007 M. Expression of the data as yields relative to glycine may be accounted for only partially by this concentration difference. To the extent that interference by a large side chain promotes by-product formation, one might expect a greater effect on yield in the presence of leucine than of glycine when the concenffation of des-glycinamide vasopressin is reduced. Possible orientations with respect to charged groups on the acvlcarbodiimide intermediate may contribute some selectivity to the nucleophilic attack. As indicated in Fig. 2, there are charged groups surrounding the reaction site. Perhaps the entering amino acid aligns itself so as to orient its hydrophobic side chain away from the charged groups of the acylcarbodiimide. Such positioning might aid in bringing the
1.63
394
~i. F. ADDY et al.
RNH-C=NHR
I O = C ~ : N t t 2CHR' Cys-Tyr-Phe-Gln- Asn-Cys-Pro -NHCH
COOH
I (Ct1214 +INH3 Fig. 2. P o s t u l a t e d final step in t h e c o n d e n s a t i o n of d e s - g l y c i n a m i d e v a s o p r e s s i n w i t h a free a m i n o acid.
hydrophilic region of an amino acid near the reaction site, the carbonyl carbon. This alignment could become most extensive and almost imperative for amino acids with large aliphatic R-groups, and higher yields could result. Whatever the details of the basis for selectivity, it must be due to the microenvironment of the reaction complex. Only in this way can the two groups of experiments be reconciled, a-N-Acetyllysine is quite small compared to des-glycinamide vasopressin. Attack of the acylcarbodiimide intermediate involving the former may be less demanding geometrically and permit higher yields of peptides. This behavior is consistent with reduced yields in nonaqueous reactions for general chemical synthesis of relatively large peptides. Acylcarbodiimides containing either desglycinamide vasopressin or N-acyllysine did not select for glycine and did not react with glycinamide at all. These results do not negate the hypothesis that prebiological peptide syntheses could have produced peptides ultimately possessing biological activity. Nor would one expect chemical evolution to be so specific as to provide only peptides eventually used for biological purposes. The present results suggest that geochemical events possessed some order but were flexible enough to provide raw materials from which substrates were selected for the evolution of life. Trends observed in the present experiments are the reverse of contemporary biochemical phenomena. If they are related to elucidation of primordial events, it may be to indicate that prebiotic synthesis was N to C as is now observed on the ribosome, not the C to N condensation employed in this study. The next experiment of this type might profitably be attempted substitution of the N-terminal residue of a biopolymer with a series of amino acids. Vasopressin with its N-terminal half-cystine would not be an appropriate starting point for such an investigation. ACKNOWLEDGMENT
This work was supported in part by and approved on April IO, 1972 f,,r publication as paper No. 418o of tile journal series of the Pennsylvania Agr;.cultural Experiment Station. REFERENCES I Borsook, H. (1954) in Chemical Pathways of~/letabolism (Greenberg, D. M., ed), Vol. 2, pp. 173222, A c a d e m i c Press, New Y o r k
MODEL FOR PRIMORDIAL PEPTIDE SYNTHESIS
395
2 Bodanszky, M. and Ondetti, M. A. (1966) Peptide Synthesis, pp. 119-122, Interscience, New York 3 Steinman, G.: Kenyon, D. H. and Calvin, M. (1966) Biochim. Biophys. Acta 124, 339-35 o 4 Steinman, G. and Cole, M. (1967) Pvoc. Natl. Acad. Sci. U.S. 58, 735-742 5 Steinman, G. (1967) Arch. Biochem. Biophys. 119, 76-82 6 Pattee, H. H. (196I) Biophys. J. I, 683-7Io 7 DeLange, R. J. and Smith, E. L. (1971) Ann. Rev. Biochem. 4 o, 279-314 8 Lucas-Lenard, J. and Lipmann, F. (1971) Ann. Rev. Biochem. 4 o, 409-448 9 Bailey, J. L. (1967) Techniques in Protein Chemistry, 2nd. edn, pp. I89-I9O, Elsevier, London io Manning, M., Wuu, T. and Baxler, J. w . M. (1968) J. Chromatogr. 38, 396-398 I I Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 12 Carnegie, P. R. and Synge, R. L. M. (1961) Biochem. J. 78, 692-696 13 Baumler, J. and Rippstein, S. (1961) Helv. Chim. Acta 44, 1162-1164 14 Meister, A. (1965) Biochemistry of the Amino Acids, 2nd. edn, p. 28, Vol, i, Academic Press, New York
BBA36318 SELECTIVITY IN AQUEOUS PEPTIDE SYNTHESIS BY I_ETHYL_3_(3-DIMETHYLAMINOPROPYL) CARBODIIMIDE A MODEL FOR PRIMORDIAL PROCESSES
MARIAN E. ADDY*, GARY STE1NMAN** ANI~ M. F. MALLETTE
Department of Biochemistry, The Pennsylvania State University University Park, Pa. 168o2
(U.S.A.) (Received June 23rd, 1972) (Revised manuscript received October 24th, 1972)
SUMMARY
Commercial vasopressin was purified, the terminal glycinamide removed by tryptic hydrolysis and the resulting des-glycinamide vasopressin was isolated and employed as a model in studying selectivity in peptide bond formation. Water soluble i-ethyl-3-(3-dimethylaminopropyl) carbodiimide was used to condense various individual aliphatic amino acids to the C-terminal end of des-glycinamide vasopressin forming analogs of 81ysine-vasopressin. Thin layer chromatography on Silica Gel G, paper chromatography and paper electrophoresis were combined to isolate peptide products. They were characterized by end-group analysis and tryptic hydrolysis. There was a general increase in yield with size of the aliphatic side chain. Although glycinamide occupies the carboxyl terminal position of lysine vasopressin, this amino acid derivative did not condense with des-glycinamide vasopressin. Nor did either of two other amides. Moreover, glycine condensed to a lesser extent than did the other amino acids used. In addition, a-N-acetyllysine was used as a second model acceptor for amino acids, this time representing the penultimate amino acid residue of vasopressin. It showed a preference for glycine over some other amino acids. The limited selectivity observed in the various condensations is attributed to the microenvironment at the reaction center, since the amino acids studied were chemically similar in their side chains and included glycine, sarcosine, alanine, aaminobutyric acid, valine, norvaline, leucine, isoleucine, and norleucine. It is suggested that prebiological synthesis of peptides by means of such condensation reactions led to somewhat higher concentrations of preferred ploducts, some of which were active in developing biological systems. * Present address: Department of Biology, Federal City College, Washington, D.C. 20001, U.S.A. ** Present address: Ames-Yissum Ltd., Shattner Center Building 3, Jerusalem, Israel.
386
M.E. ADDY et al.
INTRODUCTION
Chemical evolution concerns geochemical synthesis of molecules essential to life and focuses attention especially on nucleic acids, proteins and their precursors. Prebiological synthesis of proteins involves formation of peptide bonds in the absence oI the system presently used, based on enzymes, ribosomal assembly, and sequence information in messenger RNA. The formation of a peptide bond is a dehydrating condensation with a free energy requirement of about 4 kcal/mole (ref. i). This energy barrier is overcome biologically by utilization of energy transfer intermediates. Under primordial conditions, energy might have been obtained by coupling peptide bond synthesis with an exergonic hydrolysis which provided the necessary free energy. Carbodiimides are highly tea ctive condensing agents ~. Yet they could have been present in primordial times as isomers of protonated cyanamide anion 3. Selective incorporation of amino acids in such a system could have led to peptides and proteins with primitive biological functions. Evidence supporting this possibility has been obtained 4 from dipeptide syntheses under simulated primitive earth conditions. These dicyanamide generated dipeptides had compositional frequencies corresponding 4 to the occurrence of such dipeptides in present day proteins. It has been suggested 5 that selectivity in chemical synthesis of peptides is due to differences in side chains of the amino acids and led to some orderliness during prebiological peptide synthesis. The present investigation attempts to evaluate some of the factors which might have been responsible for this postulated orderliness. Because of its availability and convenient chemical features, the peptide hormone ~lysinevasopressin was taken as a starting point. The concept underlying this portion of the work is shown diagramtically in Fig. I. In this approach the terminal glycinamide residue was removed by tryptic hydrolysis yielding an octapeptide called desglycinamide vasopressin. Condensation of this peptide with simple aliphatic amino acids was studied as an indicator of preferential amino acid incorporation. Some selectivity in the condensation would be expected because of the proposaP that any peptide at a given state of growth may have enough chemical information to favor conditionally tile addition of particular amino acids. Moreover, the carboxylterminal amino acid of the peptide by its special position would be expected to influence the condensation by selectivity with respect to the next amino acid added. Thus, there should be two factors cooperating, the growing peptide as a whole and the carboxyl-terminal amino acid. In the present study, an attempt is made to distinguish between these two factors by employing both des-glycinamide vasopressin and a lysine derivative representing the penultimate amino acid in vasopressin, a-N-Acyllysine was used as the lysine derivative resembling as closely as possible the lysine residue in des-glycinamide vasopressin. Preliminary experiments revealed that glycinamide did not condense with desglycinamide vasopressin in the reaction system used here, nor did it condense with aN-acetylysine (Addy, M. E., Steinman, G. and Mallette, M. F., unpublished results). Therefore, tree amino acids were used in place of their amides in this study. While such a choice of reactants does not take account of present day genetic coding for glutamine and asparagine, it can be justified by absence of DNA coding for any other amides or for glycosyl groups in glycoproteins. Presumably, amino acids are incorpo-
MODEL FOR PRIMORDIAL PEPTIDE SYNTHESI~
387
rated first in free form and derivatized later. Thus, amidation of glycine to form vasopressin after first condensing the amino acid may be analogous to acetylation of the e-amino group of lysine observed 7 for certain histones or the removal of formyl groups from terminal N-formylmethione followed by hydrolytic cleavage of one or two residues from the precursors of many bacterial proteins s. MATERIALS AND METHODS
Free amino acids, I-dimethylaminonaphthalene-5-sulfonyl choride (dansyl chloride) and qysine-vasopressin were obtained from Sigma Chemical Co. a - N Acetyllysine was purchased from Calbiochem, a-N-tosyllysine from International Chemical and Nuclear Corp., and I-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride from Cyclo Chemical Co. Trypsin twice crystallized was obtained from Mann Research Laboratories. Sephadex G-I5, particle size 4o-12o, from Pharmacia Fine Chemicals, and [1-1*C]leucine, [i-14C]glycine and [I-l~C]valine were obtained from International Chemical and Nuclear Corporation and Tracerlab. Dipeptides of a-N-acyllysine and aliphatic amino acids were synthesized as follows. Each reaction mixture consisted of o.I ml of 0.06 M a-N-acetyllysine or a - N tosyllysine in o.I M HC1, o.I ml of 0.06 M amino acid in water from the list following and o. I ml of o. I M I-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The amino acids used individually were glycine, alanine, sarcosine, valine and norvaline, leucine, isoleucine, and norleucine. The carbodiimide was added last, and the reaction mixture held overnight at room temperature. In other experiments, 14C-labeled amino acids were mixed with their 12C forms before combination with other reactants. The radioactive amino acids then served as quantitative and qualitative control measures of reaction of unlabeled products. Des-glycinamide vasopressin was prepared as follows. Commercial vasopressin was relatively impure with an activity of about IOO I.U./mg, compared to about 5o0 I.U./mg for pure material. Vasopressin in water was added to trypsin and buffer solutions in a volume ratio of 3 :I :26. The buffer was 0.046 M Tris-HC1 (pH 8.1) and contained o.oi15 M CaCI~. Trypsin was in o.ooi M HC1 and was in a weight ratio to vasopressin of 1:50. Hydrolysis was continued for 24 h at 30 °C and stopped by addition of a small volume of I M HC1. The hydrolysis mixture was lyophylized and a small sample of the residue taken up in o.I M NaHCOs. This solution was used to identify products in the hydrolysate using the dansylation procedure of Bailey 9 for determination of end-groups. Dansyl derivatives were separated by thin-layer chromatography on silica gel G using the upper layer formed after equilibration of diisopropyl ether-2-butanone-acetic acidwater (8:2:5:5, by vol.). The rest of the lyophylized material was taken up in 50% acetic acid. Des-glycinamide vasopressin was isolated from this solution and purified by gel filtration according to the procedure of Manning et al. ~o developed for purification of oxytocin. Tyrosine-containing peptides from the column were located by a modified Lowry method n. Fractions containing such peptides in the major peak were pooled, diluted with 2 vol. of water, lyophylized to dryness and the residue taken up in 0.5 ml of 0.2 M acetic acid. This solution was applied to the same Sephadex column previously re-equilibrated with 500 ml of 0.2 M acetic acid. Fractions were again checked for peptide con-
3 H8
M . E . ADDY ,.'{ ct[.
tent by the modified l.owrv method after addition of an equal volume of (t.2 M NaOH. "File only majot component, believed to be tile desired product, was diluted with e vol. of water and lyophylized. File white fluffy peptide was analyzed for its amino acid content alter hydrolyzing it as follows. Approx. o.o5 mg was dissolved in o.5 ml of () M H i l , sealed in glass under Ne, and heated for ~2 h at IIO :('. The hvdrolvsate was diluted with about 5 vol. of water, evap~rated to dryness iH ~'acem, and taken up in a small volume of water, This solution is chromatographed on Whatman N~. i sheets using /er/dmtanol 2-butanone formic a(id \rater (4o:3o:i5:i5, by vo].), i-butanol acetic acid- water (8o :2o: 2o, by vol.), and phenol-water (8o :2o, w/v). Papers were sprayed with o,42;, ninhvdrin in absolute ethanol and colors developed for about 2 rain at H o "C. The amino acids of vasopressin were included as standards in their free forms. In addition, two dimensional paper chromatography, was en~ployed using the phenol-water and >butanol-acetic acid water systems. Analogs of vasopressin were syntbesized from des-glycinamide vasopressin by dissolving the latter in water to approx. ().oI M and adding o.oe5 ml to o.oI5 m! of o.o5 M solution of an amino acid. To this mixture was added ,.~.oi ml of a solution of that same amino acid labeled with 1~('. Then o.o2 5 ml of o.t M I-ethyl-3-(3-dimethylaminopropyl) earbodiimide was added and the reaction mixture held at room temperature for 4 11. I,ow voltage paper electrophoresis on \,Vhatman No, z paper with borate buffer *e at pH 9.2 was used to fractionate condensation mixtures containing radioactive components. After applsdng 3oo V for I2 h, paper strips were dried, and radioactive materials were located by exposure to X-ray fihu for I2 7 ~ h. Reaction mixtures were further fraetionated by paper chromatography as described above and bv thin layer chromatography on o.2 5 mm of Silica Gel (; on glass plates. Reaction mixtures and accompanying standards, were developed on the thin-laver plates at room temperature for about 4 I1 with I-butanol acetic acid water ({%:_m:e~;, by x~d.) or /crt-butanol--2-butanone water NH4()tl (4o:3o:2o:~o, by vol.). After air drying, separated components were detected bv ninhvdrin spray. Both chromatographic sheets and thin-layer plates containing radioactive materials were examined by exposure to X-ray film for I2 7 2 h after covering the plates with Saran \Vrap. After larger scale syntheses and separations, materials thought to be peptides were eluted and c(mcentrated to small volumes for analysis. Dipeptides isolated from condensation systems by thiu-layer chrmnatography and paper electrophoresis were studied by dansylation' and acid hydrolysis. Products of hydrolysis were separated by thin-layer c}~romatograpby and identified by cochron> atography with standard amino acids. Dansvl derivatives were identified by cochromatography with dansyl derivatives prepared from individual components used in the synthesis. In agreement with another study (Addy, M. E., Steinman, G. and Mallette, M. F., unpublisiled results) e-N-aminoacyllysine dipeptides were not (ibserved. Rather, the condensations involved the ('-terminus of (,-N-acetyllysine. Analogs of vasopressin were isolated by paper electrophoresis using X-ray film to locate them. Each was hydrolyzed by trypsin and the liberated amino acid identified by thin-layer chromatography of the hvdrolvsate and eochromatography with the corresponding reference amino acid. Vasopressin analogues and des-glyeinamide vasopressin were also detected b \ an acidic palladium chloride spray specific ~a fro_ oxidizable sulfur. Ninhydrin spray was employed for detection of components in the absence of radioactivit\.
MODEL FOR PI~IMORDIAL PEPTIDE SYNTHESIS
389
Peptides not containing radioactive amino acids were quantitated by densitometry of ninhydrin-positive components on thin-layer plates with a Photovolt nmltiplier photometer Model 52o-A with stage attachment for the plates and a Varicord Model 423 recorder. Peak area was used as a measure of peptide weight. This method measures the dipeptide as a function of the intensity and size of colored spots produced on thin-layer plates by ninhydrin spray. If the wavelengths of maximum absorption by the colored product differ with the amino acid, somewhat different color intensities would be expected at equal concentrations and any one wavelength. In this instrument the distribution of wavelengths is constant. Therefore, the data were normalized by dividing areas under peaks due to dipeptides by corresponding reference areas for the amino acid present in an individual peptide. This procedure assumes that condensation does not shift the wavelength of maximum absorption. Thus, data reported after ninhydrin treatment are not actually yields because of this procedure. Moreover, there is a contribution to color by the a-N-acyllysyl group. Since this moiety was present i n all peptides assayed in this way, the results are comparable for related condensations. Radioactive peptides on paper were determined by cutting out zones located with X-ray films and counting in Packard liquid scintillator with a Beckman Model LS-2ooB scintillometer. Data are also reported as yields relative to glycine (a-aminobutyric acid in one table) to make more manifest the trends in incorporation of amino acids. RESULTS AND DISCUSSION
a-N-Acetyllysine was condensed in groups of six different reaction mixtures with individual amino acids from the following series: a-aminobutyric acid, valine, norvaline, leucine, isoleucine and norleucine. Glycine, alanine and sarcosine form dipeptides which did not separate from the corresponding flee amino acids in the chromatographic systems used. Therefore, these three amino acids also were condensed with a-N-tosyllysine. Presence of this acyl group permitted chromatographic separation of products and residual reactants and made yield comparisons possible. Furthermore, for evaluation of any effects of the two different protecting groups on yields of dipeptides, radioactive glycine, valine and leucine were condensed with a-Nacetyllysine. The products were successfully isolated by electrophoresis and assayed by determining the radioactivity. Results from these three different sets of syntheses are presented in Tables I, II and III. The data of Table I indicate similar Yields for a-N-tosyllysylglycine, a-Ntosyllysylsarcosine and a-N-tosyllysylalanine. Therefore, condensation of a-N-acetyllysine with glycine should yield product to about tile same extent as for a-N-acetyllysylleucine in accord with the absolute yield data of Table II. On the other hand, comparisons of yields for the dipeptides of glycine in Tables I and II and of leucine in Tables II and III show that the acetyl and tosyl protective groups did make a difference in yield. Data on yields relative to giycine may be compared from the first three tables for the nine amino acids employed. The maximum difference (between norvaline and norleucine) in relative yield is about 65 %. General trends in selectivity during synthesis of a-N-aeetyllysyl dipeptides of of aliphatic amino acids are summarized as follows.
39 °
M.E. ADDY el al.
"FABLE I Y I E L D S OF ( 2 - ~ - T O S Y L L Y S I N E D I P E P T I D E S
R e a c t i o n s as in T ab le 11 [ e x c e p t t h a t a - N - t o s y l l y s i n e r e p l a c e d (z-N-acetyllysine. D a t a are m e a n s of s e v e n r e p l i c a t e e x p e r i m e n t s (eight for sarcosine).
(;lycine
.~;arcosine Alanine
18, l i .5 o I.OO
17. 7 1.14 o.98
R e l a t i v e y ield Standard deviation Yield r e l a t i v e to g l y c i n e
t8,i 1.75 i.oo
T A B L E I1 YIELDS OF RADIOACTIVE Cl-N-ACETYLLYSYLIIlPEPTIDES
R e a c t i o n s as in T a b l e I I I e x c e p t t h a t t r a c e r a m o u n t s of [I-14C]-labeled a m i n o a c i ds were a d d e d a l o n g w i t h t h e c o r r e s p o n d i n g u n l a b e l e d forms. R a d i o a c t i v i t y w a s d e t e r m i n e d in 5 ° p l of r e a c t i o n m i x t u r e before a n d a f t e r f r a c t i o n a t i o n to isolate t h e d i p e p t i d e s . Y i e l d s w e re c a l c u l a t e d as (14C in dipeptide/14C in r e a c t i o n m i x t u r e ) × IOO a n d are r e p o r t e d as t h e m e a n s of four r e p l i c a t e experiments.
Mean y i e l d (°/o) Standard deviation Y i e l d r e l a t i v e to g l y c i n e
Glvcine
Valine
Leucine
13. 4 o, 14 t,oo
II. 4 o. 23 0,8 5
14. 4 o. i i 1.o8
TABLE llI P E P T I D E Y I E L D S R E L A T I V E TO G L Y C I N E
E a c h r e a c t i o n m i x t u r e w a s o.o2 M a - N - a c e t y l l y s i n e , o.o33 M HC1, o.o2 M a m i n o a c i d a n d o.o33 M i - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l) c a r b o d i i m i d e . H e l d o v e r n i g h t a t r o o m t e m p e r a t u r e . D a t a are r e l a t i v e p e r c e n t a g e s o b t a i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s a n d a re m e a n s of s e v e n rep l i c a t e e x p e r i m e n t s . Y i e l d s r e l a t i v e to g l y c i n e are c a l c u l a t e d be l ow f r o m v a l u e of T a b l e I I for leucine usin g t h e r a t i o of 1.o8/I.42.
Relative yield Standard deviation Y i e l d r e l a t i v e to a - a m i n o b u t y r i c acid Y i e l d r e l a t i v e to g l y c i n e of T a b l e s I [ and III
a-Aminobutyric acid
Valine
Norvaline
Norleucine
Leucine
Isoleucine
lO.6 o.71
lO.8 o.7o
io.i 1.13
16.6 o.66
15.o o.73
12.o o.5o
i.oo
1.o2
0.95
1.57
1.42
I,I3
0.76
0.78
0.72
1,2o
1.o8
0.86
Except for the three smallest amino acids, glycine, alanine, and sarcosine, yield of dipeptide tended to increase with size of the side chain (Table III). Moreover, relatively high incorporations of glycine, alanine, and sarcosine as inferred by comparisons in Tables I and U with data for leucine suggest a second kind of size effect. Perhaps the kinetic mobility of the smallest amino acids permits more frequent collisions and leads to higher yield. Since all the amino acids used possess inert side chains, the selectivity among them cannot be due to chemical reactivity of the side chains. Rather, it must be due to size and geometry. Comparison of yields obtained from leucinc, isoleucine, and
MODEL FOR PRIMORDIAL PEPTIDE SYNTHESIS
391
norleucine e m p h a s i z e s t h e possibility for a role of side chain g e o m e t r y . The m e c h a n i s m p r o p o s e d 3 for this t y p e of c o n d e n s a t i o n suggests t h a t t h e i n t e r m e d i a t e for t h e last s t e p is t h e s a m e in all syntheses. I t is t h e p r o d u c t of a - N - a c y l l y s i n e a n d condensing agent. Therefore, diffeI ences in yield should be due to a m i n o acids a d d e d C-terminally. A p a r t from a n y differences in i n h e r e n t b a s i c i t y of the amino groups condensing, the y i e l d should d e p e n d also on t h e accessibility of these groups for t h e nucleophilic d i s p l a c e m e n t reaction. A n y crowding a r o u n d the amino g r o u p should lower the yield. I n t h e series norleucine, leucine a n d isoleucine, crowding increases in this order, a n d yield decreases in t h e same order as shown b y T a b l e I I I . The r e l a t i v e yields of d i p e p t i d e s show t h a t some s e l e c t i v i t y is expressed b y a - N acetyllysine as the p e n u l t i m a t e amino acid of vasopressin. I t is modified b y p h y s i c a l factors of t h e a m i n o acids condensing into t h e C-terminal position. This s e l e c t i v i t y is n o t t h e s a m e as t h a t o b s e r v e d 4 in o t h e r similar d i p e p t i d e syntheses. P r e s u m a b l y the charge on lysine is responsible for the difference in b e h a v i o r c o m p a r e d to g l y c y l dipeptides. I n these different b u t r e l a t e d systems, t h e r e was a t r e n d 4 t o w a r d r e d u c e d i n c o r p o r a t i o n into d i p e p t i d e s as a m i n o acids increased in size. TABLE IV YIELDS
OF
PEPTIDES
FORMED
BY
CONDENSING
AMINO
ACIDS
C-TERMINAL
TO DES-GLYCINAMIDE
VASOPRESSIN
The reaction mixture was 0.o 5 M in amino acid, o.oi M in des-glycinamide vasopressin, o.I M in I-ethyl-3-(3-dimethylaminopropyl) carbodiimide and o.i M in HC1. Initial pH was 2.5 and reaction time 4 h. Yields were calculated as in Table II except that the product reported was [I4C]vasopressin analog. There were five replicates of the leucine experiment, four of each of the others.
Mean yield (%) Standard deviation Yield relative to glycine
Glycine
Sarcosine
Leucine
2.54 o. 16 1.oo
1.32 0.o 5 o,51
3.6o o.22 1,42
TABLE V YIELDS OF PEPTIDES VASOPRESSIN
FORMED
BY
CONDENSING
AMINO
ACIDS
C-TERMINAL
TO DES-GLYCINAMIDE
The reaction mixture was that of Table IV except that des-glycinamide vasopressin was o.oo7 M. Yields calculated as in Tables I I I and IV, The glycine and leucine experiments were in three replicates, the others in five.
Mean yield (%) Standard deviation Yield relative to glycine
Glycine
Alanine
Valine
Leucine
Isoleucine
1.o5 0.o9 i,oo
1.29 o.o6 1,23
1.41 o.02 1.34
1.85 o.o 7 1.76
1.72 0.02 1.63
Yields are given in Tables I V a n d V for condensation o f des-glycinamide vasopressin w i t h v a r i o u s flee a m i n o acids. Different p r e p a r a t i o n s of des-glycinamide vasopressin were used for t h e e x p e r i m e n t s o f the two tables. I n general, yields o f the n o n a p e p t i d e s were low c o m p a r e d to yields in d i p e p t i d e syntheses, even t h o u g h the r e l a t i v e a m i n o acid c o n c e n t r a t i o n was higher. These lower yields would be e x p e c t e d because the size of d e s - g l y c i n a m i d e vasopressin should m a k e it react more slowly
302
M.V. AI)I)Y el ~1,
S
S
I
I
Cys-Tyr-Phe- Gin Asn-Cys -Pro Lys-GIyNH2 Lysine-vasopressin I Tryptic hydrolysis S
S
I
I
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys+ GlyNH2 Des-glycinamide vasopressin
RCHCOOH(NH2) NH2 + l-ethyl-3-(3-dimethylaminopropyl)carbodiimide Vasopressin analog Fig. i. Formation of des-glycinarnide vasopressin and its utilization in peptide bond synthesis Experimental details are given in Materials and Methods. Free amino acids were condensed with the carboxy terminal lysine residue. Attempted condensations with amino acid amides were unsuccessful.
with condensing agent than did a-N-acyllysine. Perhaps more influential, yields ol vasopressin analogs may be significantly reduced by a tendency for the condensation to occur N-terminal with des-glycinamide vasopressin, thereby attaching the new residue at the available N-terminal position. Information on the extent of this reaction would be a useful adjunct in interpreting the geometric effects on selectivity in the formation of vasopressin analogs. A general comparison of the yields of Tables IV and V suggests a strong concentration dependence. \Vhile different preparations of des-glycinamide vasopressin were used in these two experiments, this difference should not be an important factor because of the extensive isolation-purification procedure employed. Because of the limited supply, octapeptide concentration was reduced from o.oi to o.oo7 M. This change might lower materially the yield of vasopressin analogue while at the same time increasing yields of such side-products as the decapeptide with amino acid condensed to both ends of des-glycinamide vasopressin. With amino acid and condensing agent concentrations unchanged, by-products should increase rapidly in yield as concentration of des-glycinamide is reduced. Table IV reveals a lower incorporation of sarcosine than glycine. This effect might be due to steric interference by the N-methyl in condensations with the relatively large carboxyl donor, des-glycinamide vasopressin. An incoming group must approach the large acylcarbodiimide intermediate from a limited direction. The Nmethyl group of sarcosine might hinder this necessary approach. Of the amino acids examined in the experiment of Table V, incorporation into analogs of vasopressin increased in the order glycine, alanine, valine, isoleucine, and
MODFL FOR PRIMORDIALPEPTIDE SYNTHESIS
393
TABLE VI PEPTIDE
YIELDS
RELATIVE
TO G L Y C I N E
Yield data obtained according to Materials and Methods are taken from Tables I-V. They are related to glycine by dividing percentage yield by the corresponding value for glycine. Since the experiment of Table III did not contain glycine, data reduction for that table involved equalizing the value for leucine to that for leucine in Table II by dividing by the factor 13.9 calculated as the expected yield for glycine in this series from (15.o) (13.4)/(14.4) in which the factors are respectively yield of leucine dipeptide in Table III and yields of glycine and leucine derivatives in Table lI. C-Terminal Glyeine amino acid
Sarcosine
dlanine
Valine
Leucine
0.85 0.78
1.08 1.08
1.34
1.42 1.76
a-Aminobutyrate
z%Torvaline
Norleucine
Isoleucine
0.76
0.72
1.20
0.86
a-N-A cyllysyl dipeptides
N-Acyl group Tosyl Acetyl
1.oo 1.oo
0.98
1.00
Vasopressin analogues
i.oo t.oo
o.51 1.23
leucine. While glycinamide occupies this position in the natural hormone, of the amino acids with free primary a-amino groups, glycine was incorporated to the smallest extent, Moreover, incorporation increased with size of the R-group. Slightly diminished incorporation of isoleucine relative to that of leucine may be attributed to the slightly less favorable geometry of the R-group and its interference with nucleophilic attack of the amino group. It will be recalled that leucine was more extensively incorporated than isoleucine into a-N-acetyllysyl dipeptides (Table III). Preference for amino acids with larger R-groups is not likely to be due to increased hydrophobic character of larger aliphatic side chains. The reactions visualized ~ in the peptide synthesizing system are not likely to be affected by hydrophobic groups. Furthermore, if there are pK differences for these aliphatic amino acids, they are too small to be significant in these condensations. Moreover, the listed differences ~4 do not correlate with the yield differences of Table V. Table VI compares the selectivity differences discussed above for syntheses of a-N-acylysyl dipeptides with those for the larger vasopressin analogues. Data are normalized with respect to glycine to facilitate the comparison. Note that there is a maximum selectivity factor of about three in this series of aliphatic amino acids. Vasopressin analogues containing leucine were formed at des-glycinamide vasopressin concentrations of o.oi and 0.007 M. Expression of the data as yields relative to glycine may be accounted for only partially by this concentration difference. To the extent that interference by a large side chain promotes by-product formation, one might expect a greater effect on yield in the presence of leucine than of glycine when the concenffation of des-glycinamide vasopressin is reduced. Possible orientations with respect to charged groups on the acvlcarbodiimide intermediate may contribute some selectivity to the nucleophilic attack. As indicated in Fig. 2, there are charged groups surrounding the reaction site. Perhaps the entering amino acid aligns itself so as to orient its hydrophobic side chain away from the charged groups of the acylcarbodiimide. Such positioning might aid in bringing the
1.63
394
~i. F. ADDY et al.
RNH-C=NHR
I O = C ~ : N t t 2CHR' Cys-Tyr-Phe-Gln- Asn-Cys-Pro -NHCH
COOH
I (Ct1214 +INH3 Fig. 2. P o s t u l a t e d final step in t h e c o n d e n s a t i o n of d e s - g l y c i n a m i d e v a s o p r e s s i n w i t h a free a m i n o acid.
hydrophilic region of an amino acid near the reaction site, the carbonyl carbon. This alignment could become most extensive and almost imperative for amino acids with large aliphatic R-groups, and higher yields could result. Whatever the details of the basis for selectivity, it must be due to the microenvironment of the reaction complex. Only in this way can the two groups of experiments be reconciled, a-N-Acetyllysine is quite small compared to des-glycinamide vasopressin. Attack of the acylcarbodiimide intermediate involving the former may be less demanding geometrically and permit higher yields of peptides. This behavior is consistent with reduced yields in nonaqueous reactions for general chemical synthesis of relatively large peptides. Acylcarbodiimides containing either desglycinamide vasopressin or N-acyllysine did not select for glycine and did not react with glycinamide at all. These results do not negate the hypothesis that prebiological peptide syntheses could have produced peptides ultimately possessing biological activity. Nor would one expect chemical evolution to be so specific as to provide only peptides eventually used for biological purposes. The present results suggest that geochemical events possessed some order but were flexible enough to provide raw materials from which substrates were selected for the evolution of life. Trends observed in the present experiments are the reverse of contemporary biochemical phenomena. If they are related to elucidation of primordial events, it may be to indicate that prebiotic synthesis was N to C as is now observed on the ribosome, not the C to N condensation employed in this study. The next experiment of this type might profitably be attempted substitution of the N-terminal residue of a biopolymer with a series of amino acids. Vasopressin with its N-terminal half-cystine would not be an appropriate starting point for such an investigation. ACKNOWLEDGMENT
This work was supported in part by and approved on April IO, 1972 f,,r publication as paper No. 418o of tile journal series of the Pennsylvania Agr;.cultural Experiment Station. REFERENCES I Borsook, H. (1954) in Chemical Pathways of~/letabolism (Greenberg, D. M., ed), Vol. 2, pp. 173222, A c a d e m i c Press, New Y o r k
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2 Bodanszky, M. and Ondetti, M. A. (1966) Peptide Synthesis, pp. 119-122, Interscience, New York 3 Steinman, G.: Kenyon, D. H. and Calvin, M. (1966) Biochim. Biophys. Acta 124, 339-35 o 4 Steinman, G. and Cole, M. (1967) Pvoc. Natl. Acad. Sci. U.S. 58, 735-742 5 Steinman, G. (1967) Arch. Biochem. Biophys. 119, 76-82 6 Pattee, H. H. (196I) Biophys. J. I, 683-7Io 7 DeLange, R. J. and Smith, E. L. (1971) Ann. Rev. Biochem. 4 o, 279-314 8 Lucas-Lenard, J. and Lipmann, F. (1971) Ann. Rev. Biochem. 4 o, 409-448 9 Bailey, J. L. (1967) Techniques in Protein Chemistry, 2nd. edn, pp. I89-I9O, Elsevier, London io Manning, M., Wuu, T. and Baxler, J. w . M. (1968) J. Chromatogr. 38, 396-398 I I Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 12 Carnegie, P. R. and Synge, R. L. M. (1961) Biochem. J. 78, 692-696 13 Baumler, J. and Rippstein, S. (1961) Helv. Chim. Acta 44, 1162-1164 14 Meister, A. (1965) Biochemistry of the Amino Acids, 2nd. edn, p. 28, Vol, i, Academic Press, New York