PREBIOTIC CHEMISTRY

Catalysis of Glyceraldehyde Synthesis by Primary or SecondaryAmino Acids Under Prebiotic Conditions as a Function of pH

Ronald Breslow & Vijayakumar Ramalingam &

Chandrakumar Appayee

Received: 23 September 2013 /Accepted: 23 October 2013# Springer Science+Business Media Dordrecht 2013

Abstract The synthesis of an excess of D-glyceraldehyde by coupling glycolaldehyde withformaldehyde under prebiotic conditions is catalyzed by L amino acids having primary aminogroups at acidic pH’s, but at neutral or higher pH’s they preferentially form L-glyceraldehyde.L Amino acids having secondary amino groups, such as proline, have the reverse preferences,affording excess L-glyceraldehyde at low pH but excess D-glyceraldehyde at higher pHs.Detailed mechanistic proposals make these preferences understandable. The relevance of thesefindings to the origin of D sugars on prebiotic Earth is described.

Keywords Amino acids . Sugars . Amplification . Chirality . Aldol . Earth

Introduction

One of the most interesting questions about the prebiotic world has to do with homochirality—how did the L amino acids and D sugars form on Earth so polypeptides and nucleic acids couldhave well-defined structures, not as racemic or diastereomeric mixtures? We have describedhow meteoritic α-methyl amino acids, arriving on Earth with modest enantioexcesses of the Sconfiguration, can generate normal L amino acids by a decarboxylative transamination underprebiotic conditions (Breslow and Levine 2006; Levine et al. 2008; Breslow et al. 2010;Breslow 2011). We and others have shown how the resulting small excesses can be amplifiedunder either equilibrium (Morowitz 1969; Breslow and Levine 2006; Klussman et al. 2006,2007; Hein and Blackmond 2012) or kinetic (Breslow et al. 2010; Breslow 2011) conditions tohigh enantioexcesses of the L configuration in water solution. We assume that the prebioticprocesses occurred in water solution at moderate temperatures on the surface of the Earthwhere meteorites had landed.

In a preliminary publication we also described the formation of D-glyceraldehyde by aldoladdition of formaldehyde to glycolaldehyde catalyzed by amino acids (Breslow and Cheng2010). We found that all the L amino acids we examined preferentially formed an excess of D-glyceraldehyde with one exception; L-proline catalyzed the preferential formation of an excess

Orig Life Evol BiosphDOI 10.1007/s11084-013-9347-0

R. Breslow (*) :V. Ramalingam : C. AppayeeColumbia University, New York City, USAe-mail: rb33@columbia.edu

of L-glyceraldehyde. We suggested that L-proline may have been present in smallamounts prebiotically; it results from further reactions of L-glutamic acid. D-Glyceraldehyde is the likely precursor of other D sugars, building on additions toits aldehyde group, so our conclusion was that in spite of the result with L-proline itseemed likely that the D sugars were derived by catalysis from the L amino acids,which were originally derived from the meteoritic α-methyl amino acids.Glycolaldehyde is formed from formaldehyde and simple bases such as calciumhydroxide by the formose reaction (Breslow 1959) so we assume that there wereplaces where formaldehyde was present on Earth. It is difficult to imagine howsugars, polymers of formaldehyde, could have been formed without it.

We also studied the amplification of our observed modest excesses of D-glyceraldehyde by thewater evaporationmethod that was originally pioneered byMorowitz and used by him, Blackmond,and us to amplify small enantioexcesses of amino acids. We also used this technique to amplifysmall enantioexcesses of some D nucleosides to high levels of selectivity. We were able to amplifysmall excesses of D-glyceraldehyde to high levels with the same water evaporation technique.(Breslow et al. 2010). It depends on the fact that racemates are frequently less soluble thanhomochiral compounds, so evaporation of solutions with low enantioexcesses (ee’s) can concentratethem to very high ee’s as the racemates precipitate. This does not produce more of the homochiralcompound, it simply concentrates the enantiomer in excess so the solutions can contain as much as90 % or more of the L amino acid or the D nucleoside or D-glyceraldehyde along with minoramounts of the other enantiomer. Such high ee’s would presumably be enough for biology to startusing the major enantiomer preferentially.

Blackmond had been studying L-proline catalysis of other reactions under mildly acidicconditions, and saw that under basic conditions the product configuration was reversed(Blackmond et al. 2010). She more recently reported that with our conditions L-prolineafforded D-glyceraldehyde from glycolaldehyde and formaldehyde with addedtetrabutylammonium acetate, not the L-glyceraldehyde formed without it (Hein andBlackmond 2012). Thus we have now examined the synthesis of glyceraldehyde at threedefined pH regions, and with both primary and secondary chiral amino acids, to more fullyunderstand the conditions that form D glyceraldehyde and thus the higher sugars derived fromit. We find that L-proline at a measured pH of 7.6 reverses its preference to form D-glyceraldehyde 2.0±0.5 % ee, as described below. However, if the products from the otherL amino acids also reversed at higher pH, raising the pH would not bring all the amino acids,including L-proline, into line to form D-glyceraldehyde.

We have now seen that the preference for the formation of D-glyceraldehyde with catalysisby L amino acids with primary amino groups at low pH reverses to a small preference for L-glyceraldehyde at higher pHs (Table 1). We also see this reversal of stereochemistry at higherpHs when secondary L amino acids such as N-methyl-L-leucine were used as catalysts, just aswith L-proline. Herein we report the detailed study of the glyceraldehyde synthesis catalyzedby amino acids in three different pH regions.

Methods and Materials

Formaldehyde (37 % solution in water), glycolaldehyde dimer, N-methyl amino acids, aminoacids, 2,4–dinitrophenylhydrazine (stabilized with 33 % water), and HPLC grade hexane werepurchased from Sigma Aldrich. Sodium bicarbonate, sodium carbonate and HPLC gradeethanol were purchased from Fisher. All chemicals and solvents were used without furtherpurification.

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In a typical reaction at pH 2.9–4.2, 1 mmol of glycolaldehyde, 40 mmol of formaldehyde(37 % in water) and 1 mmol of the amino acid were dissolved in 16 mL of water and stirred atroom temperature in air for 3 days. At the end the pHs had dropped by a few tenths in all cases,possibly from further air oxidation of the formaldehyde. To the reaction mixture (1/6 of thetotal volume), were added 2,4-dinitrophenylhydrazine (10 mmol) and water (7 mL), and thereaction mixture was stirred at 50 °C for 8 h. The solid material was filtered away, and thefiltrate was concentrated. The crude product was subjected to successive preparative thin layerchromatographies (First solvent system: 100 % ethyl acetate, Rf=0.7; Second solvent system:5 % methanol in dichloromethane, Rf=0.4) to obtain pure glyceraldehyde 2,4-dinitrophenylhydrazone. The enantiomeric excess of glyceraldehyde hydrazone was analyzedby HPLC.

A Waters 600 HPLC system with a Waters 996 photodiode array spectrophotometer wasused for HPLC analysis. TLC silica gel 60F254 (EMD 5715-7) was used for preparative TLC.HPLC conditions: A Chiralpak AD column was used to measure the enantiomeric excess. Amobile phase solvent hexane to ethanol gradient with 0.5 mL flow rate for 120 min was usedand product dinitrophenylhydrazones (DNPs) were monitored at 350 nm. Under these condi-tions typical retention times for L-glyceraldehyde-DNP and D-glyceraldehyde-DNP are 93 and122 min respectively. Honda et al. observed E & Z isomers of glyceraldehyde hydrazones intheir HPLC conditions (Honda and Kakehi 1978). However, we did not observe mixtures ofgeometrical isomers in our HPLC conditions.

We did not use buffers since they were probably absent prebiotically, but the added aminoacids have some buffering capacity. The pH of the aldol condensation of commercial form-aldehyde and glycolaldehyde was found to be 2.9 to 4.2 depending upon the amino acids. Wehad a low pH without addition of acid since commercial formaldehyde (Sigma Aldrich252549) has a measured pH of 3.4, from the presence of formic acid produced by air oxidation.Thus we carried out the glyceraldehyde synthesis with its initial acidic pH, as previously(Breslow and Cheng 2010) and then at higher pH’s produced by addition of base. In the lowpH cases, where the interesting results were obtained, nothing extra was added to the solution,

Table 1 Amino acid catalyzed glyceraldehyde synthesis under different pH conditions

Entry Amino acids D-glyceraldehyde hydrazonea (ee %)

pH 2.9–4.2 pH 6.6–7.6b pH 8.7–9.8c

1 L-glutamic acid 34.8±1.5 –3.5±1.2 –0.4±0.4

2 D-glutamic acid –33.8d – –

3 L-aspartic acid 44.0±1.4 –1.5±0.2 –1.0±1.2

4 L-leucine 16.1±1.0 –2.9±0.6 –2.7±1.5

5 L-valine 19.0±3.1 –4.4±2.0 –3.0±0.7

6 L-proline –20.4±0.3 2.0±0.5 1.5±0.4

7 N-methyl-L-leucine –0.7±0.1 1.8±1.1 1.7±0.3

8 N-methyl-L-glutamic acid –1.8d – –

9 N-methyl-L-valine –0.9d – –

a Determined by HPLC analysis (average of 3 runs with standard deviations)b Adjusted by the addition of NaHCO3c Adjusted by the addition of Na2CO3dObtained from a single run

Catalysis of Glyceraldehyde Synthesis by Primary or Secondary Amino Acids

and the pH was generated by the small amount of formic acid in the formaldehyde. Thereactions were performed in triplicate with small standard deviations, except for three exam-ples done only once and only at an acidic pH. Of course the formaldehyde on prebiotic earthmay not have contained formic acid as an impurity, but we do not know what the pH was of asolution on prebiotic earth so we explored all three pH regions.

Results and Discussion

In agreement with our earlier results we found that all the primary L amino acids under acidicconditions (pH 2.9–4.2) produced an excess of D-glyceraldehyde (Table 1, entry 1, 3–5). Inparticular, L-glutamic acid produced D-glyceraldehyde with 34.8 % ee (entry 1). As a checkon the procedure we also used D-glutamic acid and saw a reverse of essentially the same ratio,L-glyceraldehyde in 33.8 % ee (entry 2).

When the secondary amino acid L-proline was used at low pH, L-glyceraldehyde wasformed preferentially rather than D-glyceraldehyde, with 20.4 % ee (entry 6). To establishwhether the secondary amino group explains the different behavior of L-proline in our systemwe examined a number of N-methylated L amino acids that lacked the ring structure of L-proline but also had secondary amino groups. We found that N-methyl-L-leucine, N-methyl-L-valine and N-methyl-L-glutamic acid all preferentially catalyzed the formation of L-glyceraldehyde (entries 7–9) at low pH, not the D-glyceraldehyde catalyzed without the N-methyl groups. However, proline afforded a larger ee than these other secondary amines,indicating that the cyclic character of proline also plays a role.

Then we studied the same reactions in the pH region 6.6–7.6, adjusted by the addition ofsodium bicarbonate. In this pH region the primary L amino acids produced an excess of L-glyceraldehyde, not D-glyceraldehyde, with the modest enantiomeric excesses of 1.5 to 4.4 %(Table 1, entry 1,3–5). Although the enantioselectivity is quite a bit lower than that whichresulted under acidic conditions, the reversal of chiral preference was clearly observed. WithL-proline under non-acidic conditions the major enantiomer was now D-glyceraldehyde in 2 %ee (entry 6), consistent with the magnitudes of the other ee’s at measured neutrality. When N-methyl-L-leucine (secondary amino group) was used as a catalyst at higher pHs we also sawthe formation of excess D-glyceraldehyde (entry 7) rather than the L-glyceraldehyde underacidic conditions. We did not do the higher pH studies with N-methyl-L-glutamic acid and N-methyl-L-valine.

When we performed the reactions at pH 8.7–9.8, adjusted by the addition of sodiumcarbonate, the results we obtained were very similar to the results in reactions performed atpH 6.6–7.6, but with lesser enantioselectivity.

We examined the recovered amino acid L-valine from reactions performed at both acidicand basic conditions, and saw no detectable racemization of the amino acid. We also ran thealdol reactions catalyzed by L-glutamic acid for 2 days and 4 days under acidic conditions andsaw the formation of glyceraldehyde with 35.1 % ee and 33.6 % ee respectively, indicating thatthere was no appreciable racemization of the product glyceraldehyde under those conditions.

The chiral inductions and the reversal of chiral preferences between primary and secondaryamines and with acidic versus non-acidic pHs could be explained by the following proposedmechanisms and depicted transition states (Scheme 1) modeled on those proposed by Barbas(Sakthivel et al. 2001; Ramasastry et al. 2007) for general aldol-type condensations with aminoacid catalysts. Under acidic conditions (pH 2.9–4.2) primary L amino acids condense withglycolaldehyde 1 to form the Z-enamine favored by N-H–O hydrogen bonding. Formaldehydeis brought into the Re-face of the enamine through another hydrogen bond by the carboxylic

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acid group in the transition state TS-1 that leads to the formation of D-glyceraldehyde (+)-2.With the same reaction under basic conditions, formaldehyde approaches the Si-face of theenamine due to the absence of hydrogen bonding between carboxylate group and formalde-hyde in the preferred transition state TS-3, and electrostatic repulsion by the carboxylate ion ofthe developing alkoxide anion, to give L-glyceraldehyde (−)-2.

When a secondary amino acid catalyzes the reaction of glycolaldehyde 1 and formaldehydeunder basic conditions, in the absence of hydrogen bonding in the preferred transition state TS-2, formation of the E-enamine and approach of formaldehyde through the Re-face of theenamine are directed through steric bulk for the formation of D-glyceraldehyde (+)-2. Underacidic conditions, formaldehyde approaches the Si-face of the E-enamine because of hydrogenbonding between the carboxylic acid group and formaldehyde in the preferred transition stateTS-4 that leads to the formation of L-glyceraldehyde (−)-2.

We performed our reactions under mild conditions, and to very low levels of completion toavoid further reactions of the glyceraldehyde with formaldehyde to form higher sugars. Suchhigher sugars are also formed in the simple formose reaction at high pH, where no amino acidsare present. (Breslow 1959; Delidovich et al. 2009). In our reaction with L-glutamic acidcatalyst, only 2.4 % of the glycolaldehyde was consumed after 4 days. The glyceraldehyde(20 % of the glycolaldehyde consumed) and dihydroxyacetone (13 % of the glycolaldehydeconsumed), detected and measured by HPLC as DNP derivatives, were the only significantcarbohydrate products. We found that there was no detectable loss of glyceraldehyde when itwas incubated with formaldehyde and an amino acid at low pH, so the small yield ofglyceraldehyde we found in our synthesis procedure does not reflect any loss of the product.These two products plus the recovered glycolaldehyde account for 98.4 % of the originalglycolaldehyde. We have not been able to identify any other products; in particular, we do not

Scheme 1 Proposed preferabletransition states for the formationof glyceraldehyde

Catalysis of Glyceraldehyde Synthesis by Primary or Secondary Amino Acids

see any product from Amadori reaction of glycolaldehyde with the amino acids under ourconditions, nor ethanolamine from transamination.

Weber has studied the reaction of formaldehyde with glycolaldehyde under more vigorousconditions with amino acid catalysis (pH 5.5, temperature 50 ° C), and saw a variety ofproducts derived from glyceraldehyde (Weber 2001). He observed the consumption of 89.8 %of the glycolaldehyde after 5 days. His study did not examine any chiral induction, the focus ofour work.

Conclusion

As we have shown, small excesses of D-glyceraldehyde in water can be easily amplified tohigh concentration by water evaporation, since D-glyceraldehyde is a syrup while the racemateis a less-soluble solid (Breslow and Cheng 2010; Breslow 2011). D-Glyceraldehyde can thengo on to form higher D sugars by aldol or ketol additions to the aldehyde group. Such studiesunder prebiotic conditions are underway in our laboratory, where both a ketolase and atransketolase addition reaction have been produced under prebiotic conditions (Breslow andAppayee 2013). The reactions by which meteoritic amino acids let us form L amino acids,which then lead to D sugars, are the simplest versions of the origin of homochirality on Earthfor which actual experiments under prebiotic conditions, and findings in meteorites, producesupporting evidence.

As we have pointed out (Breslow 2011), an alternative scenario is that in a firststep an α-methyl amino acid from a meteorite, with an enantioexcess of the S-isomer,could catalyze the formation of an excess of D sugars under acidic conditions, parallelto our findings, and in a second step the sugars might catalyze the formation of the Lamino acids. Pizzarello and Weber found that L isovaline (α-methyl-homoalanine)catalyzed the self-condensation of glycolaldehyde to threose with an excess of the Denantiomer (Pizzarello and Weber 2004). Thus there is evidence that the first step inthe alternative scenario is reasonable. However, there is yet no good example of thesecond step.

Blackmond has explored alternative processes to partially resolve two racemates. Inone process she performed the Sutherland reaction (Powner et al. 2009) of 2-aminooxazole with racemic glyceraldehyde and a number of L amino acids to achievea partial excess of D-glyceraldehyde (Hein et al. 2011). In the other experiment (Heinand Blackmond 2012) she performed the same Sutherland reaction with D-glyceraldehyde and racemic proline to achieve a partial excess of L-proline. Boththe Blackmond and Sutherland processes depend on the spontaneous use of a specialchemical, 2-aminooxazole, that is not known to have been present and available onprebiotic earth. This contrasts with our processes for producing L amino acids and Dsugars, where no extra reactant is needed except for formaldehyde and the α-methylamino acids that come to earth on meteorites, along with α-keto acids that can bederived from meteoritic molecules.

Our previous conclusion that the formation of D-glyceraldehyde under prebioticconditions reflects catalysis by likely prebiotic L amino acids, except L-proline, isstill correct provided the reactions occurred under acidic conditions. We have noevidence of the pH on Earth wherein this prebiotic reaction could have occurred, butconclude that if our mechanism is correct, but not necessarily otherwise, it must havebeen acidic in order to form D-glyceraldehyde guided by the predominant L aminoacids.

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Acknowledgments Support of this work by NASA is gratefully acknowledged.

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Catalysis of Glyceraldehyde Synthesis by Primary or Secondary Amino Acids