View Online / Journal Homepage / Table of Contents for this issue NJC Dynamic Article Links

Cite this: NewJ.Chem., 2011, 35, 1787–1794

www.rsc.org/njc PAPER

Hydrothermal formose reactionw

Daniel Kopetzki* and Markus Antonietti

Received (in Montpellier, France) 1st March 2011, Accepted 11th May 2011 DOI: 10.1039/c1nj20191c

The self-condensation of formaldehyde is a one pot reaction resulting in a complex mixture of carbohydrates. Based on a simple chemical, the reaction was previously considered as a prebiotic source for sugar generation. Usually, a high pH and the presence of catalytically active species are required. Here, the formose reaction was performed under hydrothermal temperatures up to 200 1C, and carbohydrates were obtained under even simpler conditions. We found no pronounced catalytic influence of active cations, and a slightly alkaline pH was sufficient to induce the reaction. Maximum yield was reached in very short times, partly less than 1 minute. No selectivity for a particular carbohydrate, although searched for, was found. Contrary to reactions performed at lower temperatures, hexoses were only formed in negligible yields, whereas the shorter carbohydrates accounted for the major fraction. Among the pentoses, ribose and the ketoses with corresponding stereochemistry were formed in higher yields compared to the reaction at lower temperature. Furthermore, we identified 2-deoxyribose in the product mix and found strong indications for the presence of other deoxy compounds. Hence, the hydrothermal formose reaction shows some remarkable differences compared to the conventional reaction at moderate temperatures.

1 Introduction Habitats with superheated water are available in submarine areas with volcanic activity and such hydrothermal environ- Downloaded by University of Oxford on 10 November 2011 As nearly 3.5 billion year old microfossils and typical isotope ments with temperatures around 200 1C at a pressure of 20 bar 1 Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C patterns in sediments indicate, life arose quite early on earth, or higher were also present at ocean sites in the hadean period.10 just about some hundred million years after the moon/earth The presented synthesis of carbohydrates is based on collision. Thus, the primary synthesis of biomolecules took formaldehyde, which can be clearly formed under hadean place at environmental conditions very different to the current conditions and is generally considered as a prebiotic molecule ones. The prebiotic chemistry community tries to resolve having contributed to the local chemistry.11 It can be synthe- the question, how complex organic molecules can be formed sized by photoreduction of CO2, but is also available via from simple precursors, and to elucidate plausible mechanisms electric discharges.12 This simple compound can be converted 2 proceeding in prebiotic environments. This is hampered by the to a mixture of different carbohydrates in a one pot reaction, lack of knowledge about the true conditions on the early earth, called the formose reaction. Under alkaline conditions and which includes the composition of the atmosphere, whether it with certain catalysts, formaldehyde polymerises to form 3 4 was neutral or reducing, or the temperature of the ocean. sugars.13 Due to the ease with which complex carbohydrates 5,6 In the famous Miller experiment, the generation of amino are synthesised from a very simple precursor, the formose acids was proven in a simple reducing atmosphere subjected to reaction has been considered to have contributed to the origin spark discharges. Recent work has mainly focused on the of life. However, due to the fast degradation of sugars and the 7 8,9 formation mechanisms towards peptides and nucleic acids, missing selectivity, this is doubtful.14–16 while the focus of this paper lies more in the formation and The kinetics of the formose reaction is quite complex, due to chemistry of carbohydrates. In this respect, we will investigate its autocatalytic nature.17 Formaldehyde usually does not whether high temperature is feasible as an energy source. react with itself establishing a carbon–carbon bond, so that the simplest sugar glycolaldehyde is formed slowly (see Scheme 1). Max-Planck-Institute of Colloids and Interfaces, Research Campus However, as soon as some condensation product is present, a Golm, D-14424 Potsdam, Germany. cascade of reactions is initiated, ultimately leading to the E-mail: [email protected]; Fax: +49 331 567 9502; formation of various straight-chain and branched carbo- Tel: +49 331 567 9538 hydrates,18,19 plus their decomposition products. Elongation w Electronic supplementary information (ESI) available: Moderate temperature experiments, NMR and GC data. See DOI: 10.1039/ of the carbohydrate backbone occurs via base-catalyzed aldol c1nj20191c condensation with formaldehyde, accompanied by isomerisations.

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 New J. Chem., 2011, 35, 1787–1794 1787 View Online

Scheme 1 Simplified mechanism of the formose reaction. In alkaline medium retro aldol reactions take place as well, A 0.5 M formaldehyde solution was heated to 200 1C under a whereupon both resulting fragments can act as initiators pressure of 100 bar. It should be noted that such a high again, thus leading to an autocatalytic system. Due to their concentration is not along with prebiotic conditions. To vary relative stability pentoses and hexoses are the main products. the pH and to probe for the potential catalysis of simple ions, For prolonged reaction times however they decompose, recogni- salts were added. Despite the fact that certain cations are sable by the yellowing of the solution. necessary at moderate temperatures, we first used inactive

Often, Ca(OH)2 is used as base because of its high catalytic sodium or potassium salts. Control experiments were conducted 2+ activity. Ca can coordinate the enediol form of carbo- at 60 1Cin0.05MCa(OH)2 or 0.1 M NaOH, respectively hydrates and thus stabilises the deprotonated species.20 (ESIw, Fig. S1–S3). Performing the reaction in NaOH in the absence of other The conversion of formaldehyde in different salt solutions is catalytically active cations does not yield any sugars. Apart shown in Fig. 1. In pure water and even under acidic conditions

from Ca(OH)2 many other catalysts of the formose reaction (such as diluted acetic acid), formaldehyde is consumed relatively have been identified.21 Naturally occurring minerals and clays, fast within a timescale of minutes. An induction period, as quite abundant in nature, can also evoke the formation of described for the formose reaction at moderate temperatures, carbohydrates when refluxing a formaldehyde solution.22–24 is not observed. With increasing basicity of the added salts, the It is even possible to induce the formose reaction photo- conversion is accelerated. Even the barely basic sodium sulfate chemically, resulting in the formation of highly branched sugar shows some effect. This trend is continued following the series alcohols.25 Apart from formaldehyde other small molecules can acetate, hydrogen carbonate and hydrogen phosphate. In a

also be employed to build up carbohydrates. Using short carbonate buffer (50 mM NaHCO3,50mMNa2CO3), the sugars, like glycolaldehyde or glyceraldehyde, other catalysts formaldehyde is consumed in less than one minute. and less harsh conditions are sufficient. Examples include zinc Of course, the fact that formaldehyde vanishes does not prolate,26 silicate27 or dipeptides,28 but in none of these cases a prove the formation of carbohydrates. In fact, the NMR Downloaded by University of Oxford on 10 November 2011 successful formose reaction using solely formaldehyde could spectra of the reactions performed in pure water, in acetic Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C be performed. acid and also when sodium acetate was added, just show the The formose reaction is usually performed at moderate Cannizzaro products formic acid and methanol (ESIw,Fig.S4). temperatures or occasionally at around 100 1C.29 In recent These solutions remained clear and colourless even for work, we have presented a continuous flow reactor which prolonged reaction times and did not show the typical yellow- allows us to perform organic reactions in water at high ing point, resulting from decomposition products when temperatures and pressures with high control and precision.30 carbohydrates were formed. This colour change was however It was for instance shown that formic acid acts under such observed for all more basic salts, indicating a successful conditions as an effective hydrogenation agent, while simple salts can take an unexpected role of being a catalyst. In the present attempt, we will apply this set-up to the formose reaction. Using simple hydrothermal reaction conditions and formaldehyde without additional initiators in the presence of only simple salts, reaction sequences are analysed. The moti- vation to study the formose reaction under such conditions is based on the lack of data on the hydrothermal behaviour of formaldehyde yielding complex molecules,31,32 but also on the fact that early earth conditions might have included various aqueous environments under similar conditions.

2 Results and discussion Effects of added salt

To establish hydrothermal conditions with high precision and Fig. 1 Conversion of formaldehyde in the presence of various salts at control, reactions were conducted in a continuous flow reactor. 200 1C and 100 bar.

1788 New J. Chem., 2011, 35, 1787–1794 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 View Online

formose reaction despite the absence of catalytically active carbohydrate formation is reached well before complete cations. In a control experiment with microwave heating, this formaldehyde consumption. yellowing occurred in glass vessels as well, suggesting that it is At moderate temperatures, the presence of Ca2+ is crucial not the reactor material which causes the observed effects. for a successful formose reaction. Even with initiator no carbohydrates are formed in the absence of catalysts. The initiator only reduces the induction period but does not Influence of an initiator and calcium ions improve the yield when the formose reaction is performed in

We also tested for the influence of both the cation variation Ca(OH)2 solution. In our experiments the total yield of and the presence of an initiator. A 0.2 M sodium acetate or, (straight-chain) carbohydrates could reach 26.5%. respectively, a 0.1 M calcium acetate solution were used to Under hydrothermal conditions however, an initiator and a investigate the effect of a potential cation catalysis. Both catalyzing counterion simply step into the competition of solutions contain the same amount of acetate and thus exhibit formose and Cannizzaro reactions. In the presence of glycol- similar basicity. Fig. 2 shows that up to two minutes reaction aldehyde the carbohydrate formation starts immediately time the consumption of formaldehyde is identical in both whereas in the absence a substantial amount of formaldehyde cases. Subsequently, the reaction accelerates in the calcium is lost before by self-disproportionation. To keep the experi- acetate solution. An interpretation could be that at this point ments limited to simple chemicals, the use of an initiator was small carbohydrates such as glycolaldehyde must have formed, omitted for further studies. The more basic salts NaHCO3 and 2+ which can specifically interact with the Ca -ions and accel- K2HPO4 or a sodium carbonate buffer also resulted in a erate the formose reaction. Testing the reaction solutions from yellowing of the solution after relatively short reaction times the first two minutes, indeed predominantly the Cannizzaro (Fig. 1) and coupled carbohydrate formation. Not only products were found in both solutions. In case of the calcium the speed of formaldehyde consumption, but also the yield salt, the solution turns yellow at later stages, and carbo- of carbohydrates was improved compared to the addition hydrates are formed. The maximum yield of carbohydrates of more neutral salts, such as Ca(CH3COO)2. This again is already reached for a formaldehyde conversion of 55%. This points to the fact that a slightly more basic pH is of is to be compared with the formose reaction at moderate greater relevance than the specific presence of Ca2+ under temperatures where the yield is at a maximum shortly after all hydrothermal conditions. formaldehyde is consumed. According to the data, decomposition of carbohydrates seems to be more accelerated at higher Typical kinetics on the example of K2HPO4 addition temperature, which is expected. The overall yield of sugars is For the model case of 0.1 M K2HPO4 addition, the kinetics of rather small. Integrating over the signals of all linear sugars formation and decomposition of carbohydrates was analysed with two to six carbon atoms, we obtain only 3.4%. The in detail. Fig. 3 shows the concentration of formaldehyde and composition and distribution of sugars will be discussed later. the products as added masses of carbohydrates with the same To compare this salt catalysis, reactions were carried out number of carbon atoms. The products were analysed by gas Downloaded by University of Oxford on 10 November 2011 with the addition of glycolaldehyde as a promoter (1 mol% chromatography as their alditol acetates. By reduction with Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C with respect to formaldehyde). This simplest sugar is a power- NaBH4 the number of species is decreased, which facilitates ful initiator of the formose reaction. Indeed, the conversion of analysis. Aldoses yield the same products as ketoses with formaldehyde is much faster (Fig. 2). Here, the sodium acetate corresponding stereochemistry. Only the linear carbohydrates solution also turns yellow, and carbohydrates are formed. In were quantified. It is known that branched sugars are also case of Na(CH3COO) the total yield is 5.1%, whereas for formed in formose reactions,18 and the GC chromatograms Ca(CH3COO)2 it reaches 12.0%. Again, the maximum of showed some weak peaks that could be attributed to them. A control experiment, in which the reduction step was omitted,

Fig. 2 Conversion of formaldehyde with addition of sodium or Fig. 3 Kinetics of formaldehyde consumption and product forma-

calcium acetate in the presence and absence of the initiator glycol- tion of a 0.5 M formaldehyde solution in 0.1 M K2HPO4 at 200 1C and aldehyde at 200 1C and 100 bar. 100 bar.

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 New J. Chem., 2011, 35, 1787–1794 1789 View Online

indicated that negligible amounts of sugar alcohols are 3 carbon atoms after loss of acetic acid and ketene.33 The ion present after reaction (ESIw, Fig. S5). This proves that there m/z = 129 is formed the same way when an additional methyl is only little cross-disproportionation of carbohydrates with group is present. Consequently, deoxysugars show this signal. formaldehyde during reaction. The induction period in 0.1 M In this GC trace also some extra peaks are present. Most

K2HPO4 is only around 25 s. During this time span about 20% importantly, we were able to quantify 2-deoxyribitol. Its of formaldehyde is lost via disproportionation. Afterwards the identity was validated by comparing the complete mass spectrum formation of carbohydrates is very rapid. It takes less than and the retention time with the reference compound. The another 30 s to reach maximum yield. maximum yield was however only 0.18 mg per 100 mg Contrary to the reaction at ambient temperatures, where formaldehyde. Still, it is worth noting that not only the true hexoses are the main products, here the shorter carbohydrates condensation products of formaldehyde are found. As the are preferentially formed. Despite the high stability that is in various peaks with the m/z = 129 ion fragment indicate, many principle expected for hexoses, their amount is insignificant at other deoxy compounds are probably formed as well. Further 200 1C. With the exception of glycolaldehyde, all different indications for the presence of deoxy species are gained from carbohydrates peak around the same reaction time. At 60 1C NMR spectra that show peaks in the region of 1–2 ppm, which

and under catalysis by Ca(OH)2 the different sugars form cannot be attributed to ordinary carbohydrates (ESIw, Fig. S6 consecutively, with the maximum yield of shorter carbo- and S7). In the control experiment at 60 1C in Ca(OH)2 we also hydrates being reached earlier than that of the longer ones. detected 2-deoxyribitol after reduction with NaBH4. Here, the This reflects a chain-like growth of the carbohydrates by maximum yield was lower (0.11 mg per 100 mg formaldehyde). successive additions of formaldehyde. The decomposition of 2-deoxyribitol is slower than that of the We speculate that under hydrothermal conditions we have a other carbohydrates. This is a general observation for all very fast equilibration between all the different compounds. compounds showing a fragment with m/z =129. Using glycolaldehyde or dihydroxyacetone as initiator did not To evaluate the amount of self-disproportionation of alter product distribution, as compared to the reaction without formaldehyde during the reaction, pH measurements were initiator. The ineffectiveness of different initiators to influence conducted after the synthesis for the model reaction in

selectivity is however well described for the formose reaction. 0.1 M K2HPO4. The variation of pH (after threefold dilution) Not only the formation, but also the decomposition is very is shown in Fig. 5. As expected the pH decreases throughout rapid. One intermediate seems to be glycolaldehyde itself, the whole reaction process. Although no sugars were formed because its concentration still increases while all other sugars in the first 30 s, the pH falls rapidly. Under the assumption diminish. The nature of the decomposition products was that the change in pH is only caused by the formation of however not further investigated. It is known that some formic acid, its concentration can be calculated with the law of polymeric species are formed. At long reaction times the mass action, taking into account the self-disproportionation solution turned dark brown and ultimately turbid and exhibited of water and the equilibria of the different phosphate species À14 a strong caramel like odour. and formic acid (with the dissociation constants Kw =10 , Downloaded by University of Oxford on 10 November 2011 Gas chromatographic analysis revealed the presence of pK1 = 2.15, pK2 = 7.20 and pK3 = 12.35 for phosphoric Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C other compounds besides straight-chain carbohydrates. The acid34 and pK = 3.76 for formic acid).35 The calculated chromatogram of the reaction at maximum yield is shown in concentration is also plotted in Fig. 5. Large amounts of acid Fig. 4. Three different ions were scanned. The signal at m/z =43 are created. The maximum concentration of formic acid originates from the acetoxy cation. Thus all compounds with reaches 100 mM, which is one-fifth of the formaldehyde hydroxyl groups show a peak in this trace. The ion m/z = 115 initially present. The Cannizzaro reaction thus accounts for is characteristic for alditols. It originates from a fragment with 40% of the formaldehyde consumption. This loss is much

Fig. 4 GC traces of the reduced and acetylated formose products for Fig. 5 Variation of pH (squares) and calculated amount of formic different ions (each scaled with different factors), for the reaction of acid (circles) for the reaction of 0.5 M formaldehyde (triangles) in

0.5 M formaldehyde in 0.1 M K2HPO4 at 200 1C and 100 bar. 0.1 M K2HPO4 at 200 1C and 100 bar.

1790 New J. Chem., 2011, 35, 1787–1794 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 View Online

higher than in the conventional formose reaction at moderate Carbohydrate selectivity temperatures. Due to the low salt concentration, the buffer It is an interesting question whether the nature of the added capacity is exhausted quite fast. When the formation of salt or catalyst had some effect on the product selectivity. carbohydrates finally takes place, the solution is neutral or Table 1 shows the maximum yield with different salts. Salts are even slightly acidic. ordered according to increasing basicity. The total yield nicely indicates that the starting pH and not any ion catalysis is the Influence of buffer capacity decisive factor. When working under hydrothermal conditions the yield is lower compared to moderate temperatures, but less The change in basicity might indeed stop the formose reaction. harsh conditions, e.g. a significantly lower alkaline pH, are To conduct the reaction under stable pH conditions, the salt necessary to induce the formose reaction. Among the hydro- concentration was raised to 0.5 M. A sodium carbonate buffer, thermal reactions, using a simple 0.1 M carbonate buffer consisting of equal proportions of Na2CO3 and NaHCO3, was resulted in the highest relative yield. Concentration and type employed. With these modified conditions the pH remained in of buffer are optimal, as the solution is initially alkaline, the alkaline range also for prolonged reaction times. Perform- allowing the formose reaction to proceed. When formaldehyde 1 ing the reaction at 200 C resulted in a dark brown solution was consumed and the maximum of carbohydrate yield already after less than half a minute. Contrary to the reactions reached, the solution became neutral. Here, sugars exhibit with low buffer capacity, the solution at high pH did not the highest stability. become turbid, even for prolonged reaction times. It appears Taking a look at various reactions performed at 200 1Cno that no carbonisation occurs in alkaline media. difference in selectivity towards a special sugar was apparent. 1 Due to the fast reaction, the temperature was lowered to 150 C. The relative amounts of carbohydrates with different carbon Here, kinetics is comparable to the system with 0.1 M numbers are roughly identical. As discussed earlier, the most K HPO at 200 1C. However, the induction period and the 2 4 striking difference is the virtually complete absence of hexoses at subsequent formation of carbohydrates are clearly more dis- 200 1C and the preferential formation of shorter carbohydrates. tinct (see Fig. 6). The formation of carbohydrates occurs quite Two different alditols, erythritol and threitol, originate from abruptly. It takes only 6 s from the beginning of their forma- the post reaction reduction of the tetroses. Both are formed in tion to maximum yield. Again, the shorter carbohydrates approximately equal amounts at 200 1C as well as at 60 1C. account for the major part, though the relative fraction of This is different regarding pentoses. Here, three sugar alcohols hexoses is more pronounced than at higher temperatures. The are identified. Under the assumption that all possible stereo- product distribution is slightly altered. In the strong alkaline isomers of pentoses are synthesised with the same probability, medium, pentoses are formed preferentially. When the buffer one should obtain 50% arabinitol and 25% of ribitol and concentration was low and the final pH slightly acidic, pen- xylitol each. Indeed, sugars yielding arabinitol account for toses, tetroses and trioses were formed in equal amounts. roughly half of the (linear) pentoses. However, we found a Besides the very fast formation of carbohydrates, their decom-

Downloaded by University of Oxford on 10 November 2011 higher concentration of ribitol compared to xylitol under position is highly accelerated in alkaline media. This is prob- hydrothermal conditions. This selectivity is reversed at 60 1C. Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C ably the reason why the overall yield was not improved by a We can speculate that this is an effect of different reactivities higher buffer capacity. Under those conditions it reaches only of the pentoses. Fig. 7 shows their fraction against reaction 7.6%. It turns out that the lower buffer capacity might even time for the hydrothermal reaction in 0.1 M K2HPO4 and the have been advantageous. At the end of the reaction the pH reaction at 60 1C in 0.05 M Ca(OH)2. In the early stages ribitol turned neutral, and sugars are more stable under these condi- dominates over xylitol in both cases. Under Ca(OH)2 catalysis, tions than at high pH.

Table 1 Maximum yield of different carbohydrates at hydrothermal and moderate temperatures of a 0.5 M formaldehyde solution in different salts, concentration of salts was 0.1 M except for Ca(OH)2 with 0.05 M

Yield at maximum [%]

200 1C, 100 bar 60 1C

NaHCO3/ Ca(OAc)2 NaHCO3 K2HPO4 Na2CO3 Ca(OH)2 Glycolaldehyde 0.95 0.80 1.41 1.20 0.55 Trioses 0.56 1.41 2.40 2.87 1.73 Tetroses 0.87 1.67 2.99 3.19 3.15 As erythritol 0.51 0.82 1.54 1.53 1.54 As threitol 0.37 0.85 1.45 1.66 1.62 Pentoses 0.90 1.46 2.48 2.94 9.17 As ribitol 0.25 0.41 0.73 0.82 1.65 As arabinitol 0.49 0.69 1.22 1.38 4.27 Fig. 6 Kinetics of formaldehyde consumption and product forma- As xylitol 0.16 0.35 0.52 0.74 3.25 tion of a 0.5 M formaldehyde solution in 0.5 M carbonate buffer at Hexoses 0.16 0.16 0.30 0.29 11.79 Total 3.44 5.50 9.58 10.48 26.50 200 1C and 100 bar.

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 New J. Chem., 2011, 35, 1787–1794 1791 View Online

nature of the additive, but not the relative product distribution. However, when the additive contains reactive sites, other reaction pathways are possible, which could push the formose reaction towards a certain product. Since the fast decomposition is the main cause for the low yield, the reaction can only be improved by either stabilising or trapping the products. We performed the reaction in the presence of 0.1 M adenine (its solubilisation was achieved by heating to 100 1C) and observed formaldehyde consumption as well as a colour change. However, no free carbohydrates could be detected and NMR spectra did Fig. 7 Fraction of pentoses for the hydrothermal reaction in 0.1 M not show any carbohydrate signals in the region of 3.5 to 4.5 ppm

K2HPO4 at 200 1C and 100 bar (a) and the reaction at 60 1C in 0.05 M (ESIw, Fig. S8). Clearly, the presence of amines blocks the Ca(OH)2 (b) showing arabinitol (circles), ribitol (squares) and xylitol formose reaction and directs it towards other pathways. When (triangles). zinc prolate was used as additive in the formose reaction, a very rapid consumption of formaldehyde took place (see Fig. 1). This salt was shown to be an efficient catalyst for aqueous aldol reactions.26 At the high temperatures used in our experi- ments, however, conversion levels of proline were similar to those of formaldehyde, indicating an incorporation of the amino acid into the products. Again, no carbohydrates could be detected. Another means of stabilisation, complexation of sugars with borate, also was not successful. Borate minerals were shown to stabilise ribose and other carbohydrates.36 When we performed

the reaction in 0.125 M Na[B(OH)4] or in a 0.125 M borate buffer prepared with borax, we observed a fast conversion of formaldehyde (ESIw, Fig. S9), but NMR spectra revealed that Fig. 8 Consumption of 0.5 M formaldehyde in 0.1 M K2HPO4 with only the Cannizzaro reaction took place. Even when 1 mol% 1 mol% glycolaldehyde at 200 1C (squares), 175 1C (circles), 150 1C glycolaldehyde was added as initiator, only marginal amounts (upward triangles) and 125 1C (downward triangles) under a pressure of carbohydrates were formed which furthermore decomposed of 100 bar. quite rapidly. Also increasing borate concentration to 0.5 M and accordingly lowering temperature to 130 1C did not result this is reversed very rapidly. Under hydrothermal conditions in significant carbohydrate formation. Obviously borate is Downloaded by University of Oxford on 10 November 2011 however, this only occurs for very prolonged reaction times, an efficient catalyst of formaldehyde disproportionation

Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C when the overall yield of sugars is already very low. In a under hydrothermal conditions and is unable to stabilise 1 neutral solution at 100 C, ribose decomposes faster than carbohydrates at elevated temperatures. Recently, silicate xylose, correlating with the percentage of free aldehyde in 14 was reported as another compound complexing and stabilising solution. Although ribose seems to be formed preferentially, carbohydrates.27 Its use was however not probed by us, as 1 its reactivity is higher. At 60 C a possible sink for ribose is the possibly occurring silica precipitation was suspected, which continued reaction to hexoses, which is negligible under would damage the employed flow reactor. hydrothermal conditions. Therefore, the fraction of ribitol does not decrease until the maximum yield of carbohydrates has been reached. 3 Conclusions The effect of temperature was investigated in the range of We have analysed the feasibility of the formose reaction under 1 1 125 C to 200 C in 0.1 M K2HPO4 (Fig. 8). Here, 1 mol% hydrothermal conditions. In contrast to the counterpart at glycolaldehyde was added to reduce the reaction time, as it is ambient temperature, less demanding conditions are required. difficult to handle extended reaction times with the employed Catalytically active species are not essential and only have flow setup. In particular we checked whether more hexoses minor effects regarding yield. A slightly alkaline solution is would be synthesised at lower temperatures. Surprisingly this sufficient to induce the reaction. In fact, depending on buffer was not the case, at least not in significant terms and in the capacity, the pH can even drop to slightly acidic at the end of analysed temperature range. Chromatograms at lower tempera- the reaction. A high pH throughout the whole reaction is in tures revealed that less side products are formed. For instance, fact disadvantageous, as product decomposition is favoured the detectable amount of 2-deoxyribitol decreased. However, above a pH too high. No selectivity towards a particular total yields were not significantly affected. product could be detected. However, considerable differences in the outcome of the reaction with respect to moderate Carbohydrate stabilisation temperatures were found. Hexoses, being the main product at low temperature, are only formed in negligible amounts.

of K2HPO4 to the formaldehyde solution, were equally valid Regarding the distribution of pentoses, we found a reversed for other salts. Only the overall yield is influenced by the selectivity towards carbohydrates yielding ribitol and xylitol.

1792 New J. Chem., 2011, 35, 1787–1794 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 View Online

Table 2 Comparison of the formose reaction under hydrothermal and moderate temperatures

High temperature and pressure Moderate temperature Only slightly basic initial conditions necessary Strongly alkaline conditions Catalytically active ions have minor effects Catalytically active ions necessary Initiator improves yield Initiator does not influence yield Induction period even without initiator quite short Initiator drastically reduces induction period Nature of initiator does not influence outcome of reaction Nature of initiator does not influence outcome of reaction Shorter carbohydrates are formed, rarely hexoses Main products are hexoses and pentoses Selectivity pentoses: arabinitol > ribitol > xylitol Selectivity pentoses: arabinitol > xylitol > ribitol Lower yield Higher yield

Apart from the true condensation products of formaldehyde, dried over anhydrous Na2SO4. Separation was achieved on a also deoxysugars were detected. Hydrothermal conditions DB-225ms column. The ions m/z = 43, 115 and 129 were seem to facilitate alternative reaction pathways. The low yield scanned, each with a dwell time of 50 ms. The ion at m/z =43 caused by rapid decomposition is a key issue. Attempts to was used for the quantification of glycolaldehyde, m/z = 129 stabilise or trap the products under hydrothermal conditions for 2-deoxyribitol and m/z = 115 for all other alditols and the have been unsuccessful so far. These findings do not provide internal standard. convincing evidence that the formose reaction played a role in Amino acids were converted to volatile products by deriva- the prebiotic formation of carbohydrates. Still, they nicely tisation with ethyl chloroformate.39 400 mL of a 4 : 1 mixture demonstrate the extent hydrothermal reactions can differ from of ethanol : pyridine were added to 600 mL of aqueous sample. moderate temperature chemistry. The aforementioned char- 50 mL ethyl chloroformate was then added and the vial briefly acteristics of the formose reaction under hydrothermal and shaken. The resulting N-ethoxycarbonyl ethyl esters were extracted moderate temperatures are summarised in Table 2. with 1 mL chloroform containing 1% ethyl chloroformate and separated on an HP-5ms column by gas chromatography. The conversion of formaldehyde was monitored photo- 4 Experimental metrically with the chromotropic acid method19 using a Perkin The experiments were performed in the X-Cube Flash Elmer Lambda 2 spectrometer. This assay is characterised continuous flow reactor from Thales Nano, which was coupled by very high sensitivity and selectivity. However, there were to a Gilson GX-271 autosampler. A 4 mL reactor made out of slight interferences with formose products, which causes the Hastelloy C-22, an alloy based on nickel, chromium and absorbance not to reach the baseline even for prolonged molybdenum, was employed. Residence time was calculated reaction times. The chromotropic acid reagent was prepared taking into account the density ratio of water under reaction by dissolving 0.5 g of chromotropic acid disodium salt dihydrate and ambient conditions. Because of the relatively low solute in 3.75 mL of water and subsequent addition of 100 mL Downloaded by University of Oxford on 10 November 2011 concentration, the density of pure water was used.37 concentrated sulfuric acid. To determine the concentration of

Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C Formaldehyde solution was prepared by refluxing an aqueous formaldehyde the sample was diluted with water to a maximum suspension of paraformaldehyde, which was purchased from of 3 mM HCHO. 100 mL were reacted with 1 mL of reagent Sigma-Aldrich. Salts and carbohydrate standards were purchased solution at 100 1C for 15 min. After cooling to room temperature from different commercial suppliers. N-Methyl imidazole, anddilutionwith5mLwatertheabsorbancewasreadoffat sodium borohydride, acetic anhydride and chromotropic acid 578 nm against a blank solution, prepared by the same protocol disodium salt dihydrate were from Sigma-Aldrich. with pure water. NMR spectra were recorded on a Bruker DPX 400 NMR. 10% Deuterium oxide was added to aqueous samples containing volatile compounds. Otherwise pure deuterium oxide was References employed as solvent. 1 J. W. Schopf, Science, 1993, 260, 640–646. Carbohydrate analysis was carried out with an Agilent 2 A. Eschenmoser, Tetrahedron, 2007, 63, 12821–12843. 6890N GC with Agilent 5975 mass spectrometer (EI ionisation) 3 G. H. Shaw, Chem. Erde: Geochem., 2008, 68, 235–264. 4 S. A. Wilde, J. W. Valley, W. H. Peck and C. M. Graham, Nature, after conversion of the sugars to their corresponding alditol 2001, 409, 175–178. acetates.38 For calibration curves, standards were prepared 5 S. L. Miller, Science, 1953, 117, 528–529. from the corresponding aldoses, except for erythritol, threitol 6 S. L. Miller, J. Am. Chem. Soc., 1955, 77, 2351–2361. and iditol, which were directly employed as such. Myo-inositol 7 B. M. Rode, Peptides, 1999, 20, 773–786. 8 R. Shapiro, Orig. Life Evol. Biosph., 1995, 25, 83–98. was added as internal standard immediately after reaction. 9 L. E. Orgel, Crit. Rev. Biochem. Mol. Biol., 2004, 39, 99–123. Afterwards carbohydrates were reduced to alditols using 10 G. Macleod, C. Mckeown, A. J. Hall and M. J. Russell, Orig. Life NaBH . Excess hydride was destroyed by addition of 500 mL Evol. Biosph., 1994, 24, 19–41. 4 11 I. Cleaves and H. James, Precambrian Res., 2008, 164, 111–118. acetic acid. Subsequently to reconcentration to about 1 mL, 12 G. Schlesinger and S. L. Miller, J. Mol. Evol., 1983, 19, 383–390. 200 mL were taken and acetylated with 2 mL acetic anhydride 13 A. Butlerow, Justus Liebigs Ann. Chem., 1861, 120, 295–298. and 200 mL N-methyl imidazole for 10 min at room temperature. 14 R. Larralde, M. P. Robertson and S. L. Miller, Proc. Natl. Acad. The solution was quenched with 5 mL water and after Sci. U. S. A., 1995, 92, 8158–8160. 15 C. Reid and L. E. Orgel, Nature, 1967, 216, 455. decomposition of excess acetic anhydride the alditol acetates 16 R. Shapiro, Orig. Life Evol. Biosph., 1988, 18, 71–85. were extracted with 1 mL dichloromethane, which then was 17 R. Breslow, Tetrahedron Lett., 1959, 1, 22–26.

This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011 New J. Chem., 2011, 35, 1787–1794 1793 View Online

18 R. D. Partridge, A. H. Weiss and D. Todd, Carbohydr. Res., 1972, 30 D. Kopetzki and M. Antonietti, Green Chem., 2010, 12, 656–660. 24, 29–44. 31 M. Osada, M. Watanabe, K. Sue, T. Adschiri and K. Arai, 19 P. Decker, H. Schweer and R. Pohlmann, J. Chromatogr., A, 1982, J. Supercrit. Fluids, 2004, 28, 219–224. 244, 281–291. 32 S. Morooka, C. Wakai, N. Matubayasi and M. Nakahara, J. Phys. 20 A. N. Simonov, O. P. Pestunova, L. G. Matvienko and Chem. A, 2005, 109, 6610–6619. V. N. Parmon, Kinet. Catal., 2007, 48, 245–254. 33 Y. Nygren, S. A. Fredriksson and B. Nilsson, J. Mass Spectrom., 21 T. Mizuno and A. H. Weiss, Adv. Carbohydr. Chem. Biochem., 1996, 31, 267–274. 1974, 29, 173–227. 34 R. N. Goldberg, N. Kishore and R. M. Lennen, J. Phys. Chem. 22 A. G. Cairns-Smith, G. L. Walker and P. Ingram, J. Theor. Biol., Ref. Data, 2002, 31, 231–370. 1972, 35, 601–604. 35 J. L. S. Bell, D. J. Wesolowski and D. A. Palmer, J. Solution 23 A. W. Schwartz and R. M. Degraaf, J. Mol. Evol., 1993, 36, 101–106. Chem., 1993, 22, 125–136. 24 N. W. Gabel and C. Ponnamperuma, Nature, 1967, 216, 453–455. 36 A. Ricardo, M. A. Carrigan, A. N. Olcott and S. A. Benner, 25 Y. Shigemasa, Y. Matsuda, C. Sakazawa and T. Matsuura, Bull. Science, 2004, 303, 196–196. Chem. Soc. Jpn., 1977, 50, 222–226. 37 E. W. Lemmon, M. O. McLinden and D. G. Friend, Thermo- 26 J. Kofoed, J. L. Reymond and T. Darbre, Org. Biomol. Chem., physical Properties of Fluid System, in NIST Chemistry WebBook, 2005, 3, 1850–1855. NIST Standard Reference Database Number 69, National 27 J. B. Lambert, S. A. Gurusamy-Thangavelu and K. B. A. Ma, Institute of Standards and Technology, Gaithersburg, MD, Science, 2010, 327, 984–986. 20899, http://webbook.nist.gov, retrieved October 15, 2010. 28 S. Pizzarello and A. L. Weber, Orig. Life Evol. Biosph., 2010, 40, 3–10. 38 A. B. Blakeney, P. J. Harris, R. J. Henry and B. A. Stone, 29 V. A. Likholobov, A. H. Weiss and M. M. Sakharov, React. Kinet. Carbohydr. Res., 1983, 113, 291–299. Catal. Lett., 1978, 8, 155–166. 39 P. Husek, J. Chromatogr., A, 1991, 552, 289–299. Downloaded by University of Oxford on 10 November 2011 Published on 16 June 2011 http://pubs.rsc.org | doi:10.1039/C1NJ20191C

1794 New J. Chem., 2011, 35, 1787–1794 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011