the emergence of primordial metabolism 
 has been postulated to play 
 a central role in the origins of life . many of the investigations so far have centered on the reductive 
 citric acid cycle . however , in his glyoxylate 
 scenario  , eschenmoser theorized 
 that glyoxylate 1 may have played an important role in 
 the development of a primordial metabolism , acting as a primal source 
 for biogenic molecules such as sugars , amino acids , and nucleobases . crucial to the scenario was the hypothetical formation of dihydroxyfumarate 
 ( dhf ) 5 , by dimerization of glyoxylic acid under the 
 influence of an umpolung catalyst such as cyanide . dhf was proposed to undergo reaction with glyoxylate and 
 aldoses , ultimately yielding biologically relevant -keto acids 
 and sugars , respectively . subsequently 
 it was shown by sagi et al . that the reaction 
 of dhf with glyoxylate , glycolaldehyde , and glyceraldehyde led , via 
 -keto acid intermediates , to the formation of triose , tetrulose , 
 and pentuloses , with remarkable efficiency . this success emphasized 
 the need to demonstrate the formation of dhf from glyoxylate . to this 
 end , we set out to investigate the cyanide - mediated dimerization of 
 glyoxylate (  glyoxoin reaction  ) with the expectation 
 of chemistry similar to the benzoin reaction . herein we report the results of this investigation : the unanticipated 
 formation of meso- and dl - tartrates 
 ( the formal reduction products of dhf ) rapidly and in high yield ( scheme 1 ) . we also present evidence that the glyoxoin reaction 
 proceeds via dhf as a putative intermediate , thus strengthening the 
 proposals made in the  glyoxylate scenario  . this facile production of tartrate combined with 
 the known dehydration of tartrate to give oxaloacetate , and its decarboxylation to give pyruvate , potentially , provides an alternate entry into 
 the citric acid cycle based on the  glyoxylate scenario  . the ( homogeneous ) 
 glyoxoin reaction of glyoxylate ( 1.0 m ) with 
 a catalytic amount of nacn ( 0.1 m ) in aqueous medium at room temperature 
 in 2.0 m naoh ( ph  14 ) showed complete consumption of glyoxylate 
 ( by c nmr after 28 h ) ; no signals corresponding to the 
 expected dhf were observed . ( high ph was required to ensure cyanide 
 was present only as its anion ; however ph 14 was obtained when 2.0 
 m naoh was prepared rather than 0.5 m. ) rather , the c 
 nmr spectrum of the crude reaction mixture ( figure 1 ) suggested the formation of meso- and dl - tartrates 8 in high yields ( in some cases exceeding 
 80% , see table s1 for example calculations ) 
 along with carbonate , oxalate 9 , formate 11 , tartronate 12 , and glycolate 13 . the 
 identities of products were confirmed by comparison to and by spiking 
 with authentic materials ( figure s1 ) . in 
 addition , the tartrates were isolated from the reaction mixture as 
 calcium salts according to literature procedure and confirmed by c nmr spectroscopy and mass 
 spectrometry ( figure s2 ) . the meso - tartrate is produced in equal or greater quantity than the combined dl - tartrates ( table s1 ) . no interconversion of meso - tartrate to and from dl - tartrates ( nor conversion to any other reaction 
 product ) was observed under reaction conditions ( figure s3 ) . reaction 
 of 
 1.0 m sodium glyoxylate ( 182/94 ppm , completely consumed ) with 0.1 
 m cyanide ( 167 ppm ) in aqueous 2.0 m naoh ( room temperature , 1 h ) 
 produces meso - tartrate 8 ( 178.9/76.1 
 ppm ) and dl - tartrates 8 ( 179.8/75.0 
 ppm ) . signals for carbonate ( 169.1 ppm ) , oxalate 9 ( 173.6 
 ppm ) , formate 11 ( 172 ppm ) , tartronate 12 ( 179.7/76.2 ppm ) , and glycolate 13 ( trace , 181.4/62.4 
 ppm ) were observed . the glyoxoin reaction 
 also produced tartrates at lower concentrations 
 ( 0.01 m of 1 and 0.002 m cn ) and at 
 lower temperatures ( 4 c ) . the reaction is , in fact , so resilient 
 that even under heterogeneous conditions ( when insoluble lithium glyoxylate 
 was substituted in place of sodium glyoxylate ) , tartrates were still 
 produced ( figure s4 ) . however , when the 
 ph was lowered ( ph  9 ) , tartrates were not observed , indicating 
 that high ph was crucial . when a control reaction ( at ph  
 14 ) was conducted by omitting the cyanide ion , no tartrates were formed ; 
 only the disproportionation of glyoxylate to glycolate 13 and oxalate 9 was observed , proceeding through the 
 well - known cannizzaro reaction ( figure s5c ) . the production of tartrates ( as opposed to dhf ) from the glyoxoin 
 reaction in basic media was unanticipated but did implicate dhf as 
 an intermediate , since tartrate is formally the reduction product 
 of dhf . therefore , a reaction of dhf 5 ( 5 mmol , insoluble ) , 
 nacn ( 1 mmol/0.1 m ) , and glyoxylic acid ( 5 mmol/0.5 m ) in aqueous 
 naoh ( 2.0 m/10 ml ) at room temperature was investigated . a vigorous 
 bubbling was observed in this heterogeneous reaction mixture for 2 
 h , at which point a c nmr spectrum of the supernatant 
 showed the production of tartrates 8 along with carbonate , 
 oxalate 9 , formate 11 , tartronate 12 , and glycolate 13 . more revealingly , when 
 the dhf / glyoxylate reaction was repeated , omitting the cyanide , tartrates 8 were still formed . when the reaction of 0.25 m of dhf 5 with 0.25 m glyoxylic acid ( in the absence of cyanide ) was 
 repeated in a mixture of 1.0 m naoh and 1.0 m lioh , the reaction mixture 
 became homogeneous ( similar to the glyoxoin reaction ) . c nmr spectrum , after 1 h , showed production of tartrates 8 , ( in 69% yield by quantitative c nmr ) with complete 
 glyoxylate conversion and little carbonate formation ( figure 2 ) ; the ratios of the accompanying product peaks 
 were similar to the glyoxoin reaction ( figure 
 s5a , b ) with higher proportion of meso - tartrate 
 ( table s1 ) . interestingly , in the heterogeneous 
 case , the c nmr spectrum of the reaction was also nearly 
 identical to the glyoxoin reaction with the exception of a much more 
 intense carbonate peak ( figure s6b ) . in 
 both heterogeneous and homogeneous cases , these results demonstrated 
 that dhf could , by itself , mediate the transformation of glyoxylate 
 to tartrates ( figure s6 ) . typical c nmr of the homogeneous dhf / glyoxylate ( without 
 cyanide ) reaction . reaction of 0.25 m glyoxylate with 0.25 m dhf in 
 aqueous 1.0 m naoh and 1 m lioh ( room temperature , 1 h ) . for nmr details while these reactions showed 
 that dhf might be an intermediate , 
 it may not be the only path in the cyanide - mediated glyoxoin reaction . tartrates could have been additionally produced from the base catalyzed 
 condensation reaction of glycolate 13 ( a product of the 
 cannizzaro reaction ) with glyoxylate 1 . this pathway 
 was ruled out , since tartrates were observed neither in the glyoxylate 
 control reaction discussed above nor when glyoxylate and glycolate 
 were combined under identical reaction conditions . this leaves dhf 
 formation and its reaction with glyoxylate as the most likely route 
 to tartrates . two possible mechanisms were considered for this formal 
 reduction of dhf 5 ( scheme 2 ) . the first is a simple cross - cannizzaro reaction in which the hydroxide adduct of glyoxylate transfers a 
 hydride to the carbonyl of the keto form of dhf leading directly to 
 tartrates 8 and oxalate 9 . the second pathway 
 involves an aldol reaction between dhf and glyoxylate resulting in 
 a six - carbon tricarboxylate 6 . this six - carbon compound 6 can undergo a hydroxide promoted fragmentation to form tartrates 8 and oxalate 9 ( scheme 2 ) . cross - cannizzaro reaction involving 
 a hydride transfer ( top ) versus an aldol - reaction followed by hydroxide - mediated 
 fragmentation ( bottom ) . to differentiate between 
 these two possible pathways , a reaction 
 of dhf and glyoxylate was conducted using c - dilabeled 
 glyoxylate . if the cross - cannizzaro hydride transfer was the sole 
 pathway , only the formation of c labeled oxalate 9 * would be expected . if the fragmentation of the six - carbon 
 tricarboxylate 6 * was the only pathway , only signals 
 corresponding to labeled tartrates 8 * would be observed . however , when c - dilabeled glyoxylic acid ( 0.1 m ) was 
 reacted with dhf ( 0.1 m ) in 1.0 m naoh and 1.0 m lioh ( homogeneous 
 reaction ) , thec nmr spectrum showed that both labeled 
 tartrates 8 * and oxalate 9 * were formed . while the presence of labeled tartrates 8 * indicates 
 that an aldol reaction fragmentation reaction must have occurred , 
 formation of labeled oxalate 9 * could be explained via 
 the competing self - cannizzaro ( as opposed to the cross - cannizzaro ) 
 reaction of glyoxylate . however , integration of the c 
 signals corresponding to the carboxylate peaks of oxalate 9 * and glycolate 13 * revealed that there is 50% 
 less labeled oxalate than labeled glycolate in the reaction mixture 
 ( figure s7c ) . this excess of glycolate 
 indicated that ( a ) there is likely some side reaction that results 
 in the production of labeled glycolate 13 * ; and ( b ) if 
 the cross - cannizzaro hydride transfer reaction is taking place , it 
 is doing so to a lesser extent than this glycolate producing side 
 reaction . therefore , although the cross - cannizzaro reaction can not 
 be ruled out entirely , this experiment supports the aldol reaction a separate reaction using unlabeled 
 glyoxylate ( 1.0 m ) and c - labeled nacn ( 0.01 m ) , clearly 
 revealed the glyoxylate cyanohydrin 2 at 125 
 ppm an otherwise empty spectral region ; as the reaction progressed , 
 a second peak appeared at 126 ppm ( attributed to the dhf cyanohydrin 4 ) persisting for 4 h before disappearing ( figure s8 ) . in the heterogeneous reaction 
 ( in 2.0 m aq naoh ) of labeled glyoxylate 
 with dhf , a second pathway was suggested by the formation of significant 
 amount ( 10% ) of singly labeled tartrates 8 ( figure s7b ) , along with singly labeled tartronate 12 . interestingly , the ( single ) labeling occurs 
 only at the carboxylate moiety of tartrates 8 and tartronate 12 as evidenced by a lack of splitting of the carboxylate signal . 
 some of this could be explained by incomplete labeling of the starting 
 material ; however , the starting material contains < 1% of singly 
 labeled material ( by c nmr , figure 
 s7a ) . this suggested that the carbon  carbon bond in 
 glyoxylate is being broken during the course of the reaction . based on the above observations , we propose an overall mechanism 
 ( scheme 3 ) , which accounts for the bulk of 
 the tartrates 8 and oxalate 9 . in this mechanism the deprotonated glyoxylate cyanohydrin 3 then 
 reacts with an additional molecule of glyoxylate to form the cyanohydrin 
 adduct of dhf 4 , which is then converted to the keto 
 form of dhf 5 ( which tautomerizes to the typical enol 
 form ) . dhf , thus formed , can react via an aldol reaction with an additional 
 molecule of glyoxylate ( c labeling shown in red ) to yield 
 a six - carbon tricarboxylate intermediate 6 * , which can 
 ( under high ph ) rearrange to 7 * via a cyanide - mediated 
 retro - aldol pathway . this mechanism accounts for the primary pathway 
 to tartrates , in which oxalate and tartrates are formed in a 1:1 ratio . these intermediates 6*/7 * can be attacked 
 at the carbonyl by a free hydroxide anion ( under high ph ) and undergo 
 fragmentation to yield tartrates 8/8 * and 
 oxalate 9/9*. this mechanism accounts for the primary 
 pathway to tartrates , in which oxalate and tartrates are formed in 
 a 1:1 ratio . additional side products are formed by pathways ( scheme 4 ) that include a series of benzoin - type rearrangements , which are possible under these high alkaline 
 conditions and which also account for the 
 formation of singly labeled products . in these pathways the tricarboxylate 
 intermediate 6(6*)/7(7 * ) can undergo benzoin - type rearrangements to a six - carbon 
 aldehyde intermediate which fragments to yield singly labeled tartrates 
 and formate . alternatively 6(6*)/7(7 * ) can undergo a retro - aldol reaction generating 
 dhf 5/5 * , which under these high - alkaline 
 conditions 
 undergoes a benzoin - type rearrangement to an aldehyde intermediate 10/10*. this intermediate can then react with 
 hydroxide and fragment to form bicarbonate , formate 11/11 * , tartronate 12/12 * , and 
 glycolate 13/13*. this second reaction pathway 
 was identified from the experiments dealing with the heterogeneous 
 decomposition of dhf alone to give formate and tartronate in 2.0 m 
 naoh . this also accounts for the presence of formate in these samples ; 
 glycolate and oxalate are also produced by the hydroxide promoted 
 fragmentation reaction of the keto form of dhf . in addition , 
 there is a side reaction that occurs when glyoxylate 1 is in a far higher concentration than dhf 5 , where 
 tartrates 8 and tartronate 12 are 
 formed in a 1:1 ratio . this side reaction ( scheme 4 , bottom ) begins at intermediate 6/7 , which decarboxylates to intermediate 14 , which then 
 undergoes an aldol reaction with an additional molecule of glyoxylate 1 to form a seven carbon tricarboxylate 15 . further 
 benzoin - type rearrangements followed by fragmentation gives rise to 
 tartrates 8 and tartronate 12 . the 
 three pathways shown above 
 account for all observed side products , including isotopically labeled 
 side products . the 
 results of the dhf  glyoxylate reaction presented here 
 are in sharp contrast to the work of sagi et al . ; there , dhf was reacted with glyoxylate in lithium hydroxide 
 at ph 78 to produce dihydroxyacetone and pentulosonic acid 
 by way of six- and seven - carbon tricarboxylate intermediates ( 6 and 15 ) via several decarboxylations . however , 
 in our work , 6 and 15 undergo attack by 
 free hydroxide at the carbonyl , yielding oxalate 9 , tartronate 12 , and tartrates 8 . while the carbonate produced 
 in the glyoxoin reaction is indicative of decarboxylations occurring , 
 no evidence was seen for any of the intermediates or products observed 
 in the work of sagi et al . ( 6 ) this contrasting 
 result demonstrates how even in consistently basic environments , ph 
 may be used to alter the product suite of this reaction , from selective 
 production of pentulosonic acid to selective production of tartrate . this dependence on ph underscores the need to investigate a wide range 
 of conditions for potentially prebiotic reactions to fully explore 
 the possibilities for producing these biologically relevant molecules . the products of this simple and robust glyoxoin reaction are stable 
 in the high - ph environment , illustrating their potential as feedstock 
 for further reactions . however two questions must be raised to determine 
 the relevance to prebiotic chemistry ; specifically , what are the abiotic 
 availabilities of glyoxylate and of a high - hydroxide concentration ? 
 for the former there are many possibilities : ( a ) glyoxylate has been 
 shown to be readily produced by photo - oxidation of acetylene under 
 anoxic conditions ; ( b ) reductive conversion 
 of carbon dioxide and carbon monoxide to glyoxylate has been experimentally 
 demonstrated ; some of these could be reasonably extrapolated to early earth scenarios ; 
 and ( c ) glyoxylate was shown by weber to form reliably in the reaction 
 of glycolaldehyde and formaldehyde , catalyzed by various primary amines , though it was not the primary product . the results of this investigation underscore the need and the opportunity 
 to further explore these options and fully investigate formation of 
 glyoxylate under prebiotic conditions . high - hydroxide concentrations 
 are considered to be extreme ( and 
 unusual ) in the conventional prebiotic line of thought . this usually 
 renders any high - ph reaction problematic in this context . however , 
 there are at least three plausible prebiotic scenarios one can consider . the first possible scenario is the widely investigated alkaline hydrothermal 
 vent system ; however , this system is not without drawbacks . related reservoirs of natural hydroxide are 
 lakes fed by alkali springs , which could 
 act as a natural reactor for this type of chemistry . while these lakes 
 may have the advantage of sidestepping the magnitude of both dilution 
 and thermolysis faced by oceanic vents , they too have their limitations . 
 another possible high - alkaline environment can be found in the interlayer 
 framework of double - layered hydroxides ( e.g. , hydrotalcites ) , which 
 are especially conducive for uptake and concentration of anions , such 
 as glyoxylate and cyanide , from dilute solutions . while each of these possibilities appear to be highly localized 
 with their attendant weaknesses in a prebiotic context , they do offer 
 potential for highly alkaline environments on early earth , that need 
 to be validated from a prebiotic perspective , for the chemistry described 
 here . notwithstanding the arguments presented above , it remains 
 that 
 the cyanide - mediated dimerization of glyoxylate at high ph to produce 
 tartrates proceeds with remarkable consistency and speed , even at 
 low temperature , low concentration , and low solubility . the fact that 
 these reactions occur reliably and that the products are stable at 
 high hydroxide concentrations demonstrates that a high ph environment 
 need not be antithetical to production of such organics in a primordial 
 setting . moreover , the use of cyanide as a catalyst is to be contrasted 
 with many other conventional prebiotic scenarios that start with cyanide 
 as a source molecule . in the context of primordial 
 metabolism , the robust production 
 of tartrates from glyoxylate opens up new venues for the origins of 
 biologically relevant small molecules . the simple and known dehydration 
 of tartrates results in oxaloacetate which 
 is known to decarboxylate to give pyruvate , thus providing an entry into a prebiotic citric acid cycle . in extant 
 biology , oxaloacetate is the entry point to the citric acid cycle , 
 reacting with pyruvate to form citrate . there are examples in extant biology where tartrate is utilized 
 and metabolized for production of oxaloacetate to be used in the citric 
 acid cycle . thus , it is reasonable to posit that tartrates could 
 have acted as a source of small molecules , which could become part 
 of an emerging proto - metabolic process ( e.g. , reductive citric acid 
 cycle ) . thus , the glyoxoin reaction ( and the  glyoxylate 
 scenario  ) could serve as a plausible alternative start for 
 rudimentary chemical evolution ( scheme 5 ) . glyoxylic acid 
 monohydrate ( 0.110 mmol , 10100 equiv ) and sodium cyanide 
 ( 0.011 mmol , 1 equiv ) were each weighed in individual 6 dram 
 vials . ten ml of 2.0 m naoh or 2.0 m lioh was then added to the vial 
 containing the sodium cyanide . the solution was then transferred by syringe to the vial 
 containing the glyoxylic acid . the cyanide solution was added down 
 the side of the vial to the glyoxylic acid over the course of 20 
 s and then closed , and the vial was cooled ( by running cool water 
 over the vial ) as necessary to avoid with excess heating . dihydroxyfumaric 
 acid dihydrate ( 0.15 mmol , 5 equiv ) , 
 glyoxylic acid monohydrate ( 0.15 mmol , 5 equiv ) , and sodium 
 cyanide ( 0.011 mmol , 1 equiv ) were each weighed in individual 
 6 dram vials . five ml of 2.0 m naoh was then added to the vial containing 
 the sodium cyanide . in 
 a separate vial an additional 5 ml of 2.0 m aq naoh was then added 
 to the vial containing the glyoxylic acid . the 
 glyoxylate solution was added down the side of the vial to the dihydroxyfumaric 
 acid over the course of 20 s and then closed , and the vial 
 was cooled ( by running cool water over the vial ) as necessary to avoid 
 with excess heating . dihydroxyfumaric 
 acid dihydrate ( 0.15 mmol , 1 equiv ) and glyoxylic acid monohydrate 
 ( 0.15 mmol , 1 equiv ) were each weighed in individual 6 dram 
 vials . five ml of 2.0 m naoh as then added to the vial containing 
 the glyoxylic acid monohydrate . this vial was mixed until no solid 
 remained . in a separate vial , 5 ml of 2.0 m aq lioh the glyoxylate solution was then transferred 
 into the vial containing the dhf solution , and the vial was closed 
 and shaken . alternately , adding dhf to glyoxylate resulted in no noticeable 
 differences .
an abiotic formation of meso- and dl - tartrates in 80% yield via the cyanide - catalyzed 
 dimerization 
 of glyoxylate under alkaline conditions is demonstrated . 
 a detailed 
 mechanism for this conversion is proposed , supported by nmr evidence 
 and 13c - labeled reactions . 
 simple dehydration of tartrates 
 to oxaloacetate and an ensuing decarboxylation to form pyruvate are 
 known processes that provide a ready feedstock for entry into the 
 citric acid cycle . while glyoxylate and high hydroxide concentration 
 are atypical in the prebiotic literature , there is evidence for natural , 
 abiotic availability of each . 
 it is proposed that this availability , 
 coupled with the remarkable efficiency of tartrate production from 
 glyoxylate , merits consideration of an alternative prebiotic pathway 
 for providing constituents of the citric acid cycle .