Energy Conservation via Thioesters in a Non-Enzymatic ...

doi.org/10.26434/chemrxiv.8832425.v1

Energy Conservation via Thioesters in a Non-Enzymatic Metabolism-likeReaction NetworkElodie Chevallot-Beroux, Jan Gorges, Joseph Moran

Submitted date: 09/07/2019 • Posted date: 09/07/2019Licence: CC BY-NC-ND 4.0Citation information: Chevallot-Beroux, Elodie; Gorges, Jan; Moran, Joseph (2019): Energy Conservation viaThioesters in a Non-Enzymatic Metabolism-like Reaction Network. ChemRxiv. Preprint.

Life’s catabolic processes capture chemical energy from the oxidative breakdown of metabolites. In thecatabolic pathways at the core of biochemistry, the oxidation of α-ketoacids or aldehydes is coupled to thesynthesis of thioesters, whose energy-releasing hydrolysis is in turn coupled to the production of adenosine5’-triphosphate (ATP). How these processes became linked before life emerged, and thus how the frameworkfor modern bioenergetics was established, is a major problem for understanding the origins of biochemistry.The structure of biochemical networks suggests that the intermediary role of thioesters in biological energyflows, and their central role in biosynthesis, is a consequence of their entry into metabolism at the earlieststage of biochemical evolution. However, how thioesters could have become embedded within a metabolicnetwork before the advent of enzymes remains unclear. Here we demonstrate non-enzymatic oxidant- orlight-driven thioester synthesis from biological α-ketoacids and show it can be integrated within aniron-promoted metabolism-like reaction network. The thioesters obtained are those predicted to be pivotal incomputational reconstructions of primitive biochemical networks (acetyl, malonyl, malyl and succinylthioesters), demonstrating a rare convergence between top-down and bottom-up approaches to the origins ofmetabolism. The diversity and simplicity of conditions that form thioesters from core metabolites suggests theenergetic link between thioester synthesis and catabolism was in place at the earliest stage of prebioticchemistry, constraining the path for the later evolution of life’s phosphorus-based energy currencies.

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Energy conservation via thioesters in a non-enzymatic 1 metabolism-like reaction network 2 3 Elodie Chevallot-Beroux,‡ Jan Gorges,‡ Joseph Moran* 4 5 Université de Strasbourg, CNRS, ISIS, 8 allée Gaspard Monge, 67000 Strasbourg, France 6 *[email protected], ‡these authors contributed equally to this work 7

Life’s catabolic processes capture chemical energy from the oxidative breakdown of 8

metabolites. In the catabolic pathways at the core of biochemistry, the oxidation of α-9

ketoacids or aldehydes is coupled to the synthesis of thioesters, whose energy-releasing 10

hydrolysis is in turn coupled to the production of adenosine 5’-triphosphate (ATP). How these 11

processes became linked before life emerged, and thus how the framework for modern 12

bioenergetics was established, is a major problem for understanding the origins of 13

biochemistry. The structure of biochemical networks suggests that the intermediary role of 14

thioesters in biological energy flows, and their central role in biosynthesis, is a consequence 15

of their entry into metabolism at the earliest stage of biochemical evolution. However, how 16

thioesters could have become embedded within a metabolic network before the advent of 17

enzymes remains unclear. Here we demonstrate non-enzymatic oxidant- or light-driven 18

thioester synthesis from biological α-ketoacids and show it can be integrated within an iron-19

promoted metabolism-like reaction network. The thioesters obtained are those predicted to be 20

pivotal in computational reconstructions of primitive biochemical networks (acetyl, malonyl, 21

malyl and succinyl thioesters), demonstrating a rare convergence between top-down and 22

bottom-up approaches to the origins of metabolism. The diversity and simplicity of conditions 23

that form thioesters from core metabolites suggests the energetic link between thioester 24

synthesis and catabolism was in place at the earliest stage of prebiotic chemistry, 25

constraining the path for the later evolution of life’s phosphorus-based energy currencies. 26

A major goal of origins of life research is to understand how and why chemistry self-27

organized into today’s biochemistry.1,2,3 The recent discovery of chemical reaction networks that 28

break or form bonds in ways closely resembling biochemistry suggests that core metabolic 29

pathways emerged from simpler non-enzymatic precursors, providing a straightforward 30

explanation for why biochemistry came to be the way it is.4,5,67,8,9,10 However, non-enzymatic 31

reaction networks that combine metabolite synthesis with life’s ability to capture chemical energy 32

from catabolic (breakdown) processes have yet to be demonstrated. 33

The hydrolysis of thioesters is highly exergonic, which allows them to occupy a central 34

position in biosynthesis and to act as life’s sulfur-based energy carriers.11 Accordingly, thioesters 35

are thought to have played a dominant role in early biochemical evolution – a concept that De 36

Duve dubbed the “Thioester World”.12,13,14 In biology, two of the main catabolic routes for 37

thioester synthesis are the oxidative decarboxylation of α-ketoacids and the oxidation of 38

aldehydes, both of which employ the thiol cofactor A (CoA), and which constitute key steps in 39

core metabolic pathways such as the Krebs cycle and glycolysis (Figure 1). Recent 40

computational predictions, based on the network structure and thermodynamics of all 41

documented metabolic reactions, also support the existence of an early metabolic network 42

hinging on thioesters.15,16 Specifically, acetyl, malonyl, malyl and succinyl thioesters were 43

predicted to emerge within or from a reaction network resembling a primitive (reverse) Krebs 44

cycle.16 Here we show that the same thioesters can be produced non-enzymatically from 45

biological α-ketoacids and integrated into a Krebs cycle-like chemical reaction network, in good 46

agreement with computational predictions about the structure of primitive metabolism.15,16 47

48

49

Figure 1 Energy conservation via thioesters in biosynthesis. A) Thioester synthesis in the Krebs 50 cycle. Oxidative decarboxylations of α-ketoacids are highlighted using red arrows. Additional 51 biological reactions, such as the aldol reaction of acetyl thioester with glyoxylate to give malyl 52 thioester, are also shown. A similar network was predicted to have been at the core of primitive 53 metabolism, by starting from simple seed molecules and applying a thermodynamically constrained 54 network expansion algorithm to grow the network using known biological reactions.16 B) Thioester 55 synthesis via aldehyde oxidation in alcohol metabolism and glycolysis. C) Nature’s most important 56 thiol, coenzyme A (CoA) and a simpler analogue used in this study, N-acetylcysteamine. 57

58

Experimental work on prebiotic thioester synthesis is limited, with the most important 59

examples arising from aldehydes17,18,19,20 or acetyl thioester synthesis from carbon monoxide and 60

methyl thiol in very low yields.21 Inspired by the synthetic organic chemistry literature,22,23 we 61

searched for simple aqueous inorganic conditions that might enable thioester synthesis from α-62

ketoacids and thiols. Our initial investigations focused on the reaction between pyruvate, the 63

prebiotically plausible biological precursor to sugars and several amino acids,4,5,6,24 and N-64

SHNH

NH

OH

Me MeO

PO

PO

O

P O-

O-O

HO

NN

NN

H2N

OO-

O

O-

O

O O

coenzyme A (CoA)

SHNH

Me

O

N-acetylcysteamine

-O

O

O

O-

O

-O

O O

O-

O

-O

O

O-

O

-O

O

O-

O-O O

OH

-O

O

SR

O

citrate

ADP, Pi

R-SH, ATP

oxaloacetate

succinate

succinyl-thioester

-O

O

SR

O-O O

OHcitryl-thioester

H2O

R-SH

Me

O

SR

Me

O

O-

O

pyruvate

acetyl-thioester

2 steps

A

B

Me

O

SR

acetyl-thioester

Me H

O

acetaldehydeOH

OP

O

OO

O

OH

OP

O

OO

O

S-enzymeNAD+ NADHR-SH HS-enzyme

NAD+ NADH

glyceraldehyde 3-phosphate

-O

O

OH O

SR

-O

O

O

H

glyoxylate

malyl-thioester

-O

O O

SR

malonyl-thioester

C

CO2ATP. ADP

Pi

NAD+, R-SH

NADH, H+, CO2

NAD+

R-SH

NADH/H+

CO2

3 steps

α-ketoglutarate

acetylcysteamine, a simpler, potentially ancestral,25 analogue of CoA. Optimization of oxidizing 65

agent, catalyst, and temperature (see Table S-1) converged on sulfate radical-generating 66

conditions identical to those reported to drive Krebs cycle-like breakdown of core metabolites5 67

(S2O82- as oxidant, FeS as catalyst, 70 °C), giving the corresponding acetyl thioester in 27% yield 68

(Table 1, entry 1, method A). The remaining material was found mostly to be unreacted pyruvate 69

and a small amount of acetate, according to GC-MS and NMR analysis (Table S-2). Thioester 70

synthesis could equally be carried out with another water-soluble thiol (Scheme S-3), or with a 71

disulfide instead of a thiol (entry 2, method A). Two other key intermediates of the Krebs cycle, α-72

ketoglutarate and oxaloacetate also underwent decarboxylative thioesterification to give the 73

corresponding succinyl thioester, an analogue of the biosynthetic precursor to many co-factors, 74

and malonyl thioester, an analogue of the precursor to biological fatty acid synthesis, respectively 75

(entries 3-4, method A). Malonyl thioester formation is accompanied by decarboxylation to give 76

acetyl thioester as a secondary product (entry 4). Malyl thioester was also formed under the 77

same conditions starting from 4-hydroxyketoglutarate (entry 5, method A). UV-A light, thought to 78

have constituted a significant portion of the solar spectrum on the early Earth,26 was equally able 79

to generate sulfate radicals from S2O82-, and therefore to deliver thioesters from α-ketoacids at 80

ambient temperature (entries 1-5, method B). Alternatively, UV-A light alone was found to trigger 81

thioester formation under acidic conditions (entries 1-5, method C). In this case however, the 82

reduced reactivity with disulfides suggests the mechanism is distinct from the light-triggered 83

reaction in the presence of S2O82- (entry 2, method C). The direct photochemical reaction was 84

found to be independent of the chemical nature of the acid (Table S-1), ruling out the 85

involvement of photogenerated sulfate radicals from bisulfate.27 Acetaldehyde, a representative 86

aldehyde, was also found to furnish acetyl thioester under thermal and photochemical sulfate 87

radical-forming conditions (entry 6, methods A-B), as well as under direct photochemical 88

conditions similar to those previously reported by Weber (entry 6, method C).17 Thus, all three 89

sets of conditions are able to generate thioesters from both α-ketoacids and aldehydes. 90

Table 1. Thioester formation from biological α-ketoacids or aldehydes and N-acetylcysteamine.

Entry Substrate Thioester Product(s) Product Yield (%) ± SD ‡

Method Aa Method Bb Method Cc

1 pyruvate acetyl thioester 27 ± 1 31.2 ± 0.4 22.1 ± 0.4

2d pyruvate acetyl thioester 34 ± 1 32 ± 2 4.9 ± 0.2

3 α-ketoglutarate succinyl thioester 10 ± 2 25 ± 2 13 ± 1

4 oxaloacetate malonyl thioester 6.7 ± 0.4 24 ± 2 8 ± 2

acetyl thioester 2.6 ± 0.3 2.4 ± 0.3 < 0.5

5 hydroxyketoglutaratee malyl thioester 2.0 ± 0.1 10 ± 1 3.5 ± 0.2

6 acetaldehydef acetyl thioester 15 ± 1 13 ± 2 20 ± 2

‡ Reported values were determined by GC-FID after an extraction procedure or by LCMS and represent the average of at least three runs. SD = standard deviation. See the Supplementary Information for additional control experiments. Unless otherwise noted, 0.5 mmol of the substrate and 0.1 mmol of the thio compound were employed. aMethod A: K2S2O8 (2.0 equiv), FeS (0.5 equiv) in H2O, 3 h, 70 °C. bMethod B: K2S2O8 (2.0 equiv), FeS (0.5 equiv) in H2O, 6 h, UV-A. cMethod C: KHSO4 (3 M) in H2O, 6 h, UV-A. d0.05 mmol of N-acetyl cysteamine disulfide was used instead of N-acetyl cysteamine. eHydroxyketoglutarate was freshly prepared from glyoxylate and oxaloacetate without isolation. f1.0 mmol of the substrate were used.

91

We recently reported an iron-promoted non-enzymatic reaction network in which most 92

Krebs cycle metabolites are formed from pyruvate and glyoxylate, including other α-ketoacids.8 93

To mimic the biological integration of α-ketoacid synthesis, α-ketoacid breakdown and thioester 94

synthesis, we simulated non-equilibrium environments where material could cycle between 95

neutral and oxidized regions, or between dark and light regions. In a first experiment, pyruvate, 96

glyoxylate, ferrous iron and N-acetylcysteamine were mixed in water at 70 °C for 1 h, after which 97

time K2S2O8 was added and the reaction continued without changing the temperature. At 4 h, 98

acetyl (1.4 mM), succinyl (0.1 mM) and malyl (3.6 mM) thioesters were detected, as well as 8 99

carboxylic acid metabolites (Table S-3). In a second experiment, pyruvate, glyoxylate and 100

ferrous iron were mixed in water at 70 °C for 1 h, after which time N-acetylcysteamine and 101

KHSO4 were added and the reaction was exposed to UV-A light at 22 °C. At 7 h, acetyl (0.1 mM), 102

succinyl (0.2 mM) and malyl (0.06 mM) thioesters were detected, as well as 5 carboxylic acid 103

metabolites (Table S-3). Thus, different types of non-equilibrium environments (neutral/oxidized 104

or dark/light) can lead to a non-enzymatic reaction network in which α-ketoacid and thioester 105

synthesis are coupled, as in biochemistry (Figure 2). 106

107

108 109 Figure 2 Thioester formation from an iron-catalysed reaction network generated from pyruvate, 110 glyoxylate and N-acetylcysteamine, achieved through redox or light cycling. Molecules in black 111 represent those observed in the presence of Fe2+, thiol and oxidant. Molecules in grey represent 112 those observed in the presence of Fe2+ only. Additional reactions and observed metabolites have 113 been omitted for clarity. 114

115

In summary, inorganic oxidants or light enable non-enzymatic thioester synthesis from 116

metabolites in a way that closely mimics energy conservation within biological catabolism, and 117

that is integrated with metabolite-generating chemical networks. Synthesis and breakdown of 118

metabolites could have been linked in an energy-conserving way from the outset of prebiotic 119

chemistry, in accord with computational models of an “onion-like” growth of metabolism from one 120

initially based on C, H, and O to one incorporating S, N, and then P.12,13,15,16 The simplicity of the 121

conditions observed here suggest thioester synthesis would have been difficult to avoid in any 122

near-surface environment where light, α-ketoacids or aldehydes and thiols were present. A 123

primitive form of bioenergetics built around thioesters therefore may have emerged very early on, 124

setting the stage for the complex life-like behaviours characteristic of thioester networks,28,29 and 125

imposing constraints on later chemical energy currencies based on phosphorous. Finally, 126

Me

O

O-

OO

O-

O

-O

O

OH

O-

O

-O

O

O-

O

-O

O

O-

O

-O

O

OH

-O

O

O-

OO-OO

-O

O

O-

O-

O2C OH

O

-O

O

O-

O

O

O-

O

-O

O

O

O-

O

OH-O

Me O-

O

OO

O-

O

OO

O-

OO

O-

acetate

pyruvate

oxaloacetate

malate

fumarate

succinate

α-ketoglutarate isocitrate

glyoxylate

glyoxylate

glyoxylate

oxalohydroxyglutarate

oxopentenedioate

hydroxyketoglutarate

O

O-

O

-O

malonate

input

SR

O

-O

O

Decarboxylative thioesterification (-CO2 - 2e- - H+ + RSH)

Hydrolysis (+H2O - RSH)

Me SR

O O

-O

O

SR

acetyl thioester malonyl thioester

succinyl thioester

SR

OOH-O

Omalyl thioester

input

hydrogen-rich geochemical environments where reductive metabolite synthesis6 could occur 127

within proximity of oxidants or light, such as surface hydrothermal vents, warrant consideration 128

for the origin of life.30 129

130

Funding: This project has received funding from the European Research Council (ERC) under 131

the European Union's Horizon 2020 research and innovation programme (grant agreement n° 132

639170) and from ANR LabEx “Chemistry of Complex Systems” (ANR-10-LABX-0026 CSC). 133

Acknowledgements: J.-L. Schmitt and C. Antheaume are gratefully acknowledged for 134

assistance with LC-MS and GC experiments. We also thank M. Coppe for help with the NMR 135

experiments. 136

Author contributions: J.M. supervised the research and the other authors performed the 137

experiments. All authors contributed intellectually throughout the study. All authors wrote the 138

paper and E.C.-B. and J.G. assembled the Supplementary information. 139

Competing financial interests: Authors declare no competing financial interests. 140

Data and materials availability: All data is available in the main text or the supplementary 141

information file. 142

Supplementary Information: 143

Materials and Methods 144

Figures S-1 to S-9 145

Tables S-1 to S-3 146

Scheme S-1 to S-4 147

References 148

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S1

Supporting Information

Energy conservation via thioesters in a non-enzymatic

metabolism-like reaction network

Elodie Chevallot-Beroux,‡ Jan Gorges,

‡ Joseph Moran*

Université de Strasbourg, CNRS, ISIS UMR 7006, F-67000 Strasbourg, France

[email protected], ‡these authors contributed equally to this work

S2

Content

1. General information ........................................................................................................................ 3

2. Analytical methods .......................................................................................................................... 5

2.1. GC-FID analysis .................................................................................................................... 5

2.2. LCMS analysis ...................................................................................................................... 7

2.3. GC-MS analysis .................................................................................................................. 11

2.4. NMR analysis with water suppression ................................................................................ 11

3. Synthetic procedures ..................................................................................................................... 12

3.1. Synthesis of starting materials ............................................................................................. 13

3.2. Synthesis of reference compounds ...................................................................................... 16

3.3. Formation of thioesters under prebiotic conditions ............................................................. 21

3.4 Thioester formation from a non-enzymatic reaction network ............................................. 27

4. Experimental data .......................................................................................................................... 29

4.1. NMR spectra of the synthesized compounds ...................................................................... 29

4.2. NMR spectra of reference compds. in H2O/D2O with DSS as IS with water suppression .. 40

4.3. NMR spectra of selected reaction mixtures showing prebiotic thioester formations .......... 48

4.4. GC-FID chromatograms of authentic samples .................................................................... 57

4.5. GC-FID chromatograms of the prebiotic thioester formation ............................................. 60

4.6. GC-FID chromatograms of the network combined with thioester formation ..................... 68

4.7. LC-MS chromatograms of authentic samples ..................................................................... 70

4.8. LC-MS chromatograms of thioester formation under prebiotic conditions ........................ 76

4.9. LC-MS chromatograms of the network combined with thioester formation ...................... 82

4.10. GC-MS chromatograms of authentic samples ..................................................................... 85

4.11. GC-MS chromatograms of the network after thioester formation ...................................... 86

5. Supporting references .................................................................................................................... 92

S3

1. General information

All reactions were carried out in oven dried glassware under an atmosphere of argon. All glassware

and stir bars were pre-washed with acid, followed by distilled water and acetone, and oven dried to

prevent any cross-contamination by metal salts. Unless otherwise noted, all reagents and solvents were

purchased from commercial suppliers and used without further purification. Water was obtained from

a Milli-Q purification system (18 MΩcm) and was purged with argon before use.

For water suppression 1H-NMR spectra were recorded on a Bruker Avance300 (300 MHz)

spectrometer at ambient temperature in a H2O:D2O mixture (6:1) as solvent, with sodium 3-

(trimethylsilyl)-1-propanesulfonate (DSS) as the internal standard (TMS peak at 0 ppm). Solvent

suppression was achieved through excitation sculpting, using the Bruker ZGESGP pulse program

adjusted for the water resonance. Integration was performed using ACD/NMR Processor Academic

Version 12.00 software.

Regular 1H- and

13C-NMR spectra were recorded on a Bruker Avance300 (300 MHz), Bruker

UltraShield 400 (400 MHz) or a Bruker Avance 500 (500 MHz) spectrometer at ambient temperature

and are reported in ppm using the solvent signal as internal reference (CDCl3 at 7.26 ppm (1H) or

77.00 ppm (13

C), DMSO-d6 at 2.50 ppm (1H) or 39.51 ppm (

13C)). Data are reported as: multiplicity

(br. s = broad singlet, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling

constants (in Hz) and integration. Melting points were obtained on a Büchi Melting Point B-450

apparatus without correction. High resolution mass spectrometry (HRMS) analysis was performed on

a Bruker MicroTOF-Q (ESI) instrument.

LC-MS analysis was performed on a ThermoFisher Scientific UltiMate 3000 UHPLC-system

equipped with an C18 ThermoFisher Hypersil Gold 10 mm column using an Exactive Plus EMR

Orbitrap detector. Acetonitrile (+ 0.1 % formic acid) and water (+ 0.1 % formic acid) were used for

the mobile phase. The column was maintained at 25 °C and the solvent gradient was used as

following: 2% MeCN for 1 min, linear gradient to 15% MeCN over 3.5 min, linear gradient to 100%

MeCN over 1 min, 100% MeCN for 2.5 min, linear gradient to 2% MeCN over 0.5 min, 2% MeCN

for 1.5 min. The analytes were quantified using a calibration of authentic samples by integration of the

peak area of a specified m/z range. Unless otherwise noted 0.1 µL of the sample solutions were

injected.

GC-MS analysis was performed on a GC System 7820A (G4320) using an Agilent High Resolution

Gas Chromatography Column (PN 19091S – 433UI, HP – 5MS UI, 28 m×0.250 mm, 0.25 Micron, SN

USD 489634H). The system was connected to an MSD block 5977E (G7036A). Hydrogen (99.999 %

purity) was used as carrier gas at a constant flow rate of 1.5 mL/min. The analysis was carried out in a

splitless mode with 1 μL injection volume, at the injection port temperature of 250 °C. The column

was maintained at 60 °C for 1 min, then ramped at 30 °C/min to 310 °C with 3 min hold, and the total

S4

running time was 12.33 min. The mass spectrometer was turned on after a 2-min solvent delay, and

was operated at the electron ionization (EI) mode with quadrupole temperature of 150 °C. Data was

acquired in the full-scan mode (50-500 amu).

GC-FID analysis was performed on a GC System GC7890B with an FID-detector using an Agilent

High Resolution Gas Chromatography Column (PN 19091S – 433UI, HP – 5MS UI, 28 m×0.250 mm,

0.25 Micron, SN USD 489634H). Hydrogen (99.999 % purity) was used as carrier gas at a constant

flow rate of 5.0 mL/min. The analysis was carried out in a splitless mode with 1 μL injection volume,

at the injection port temperature of 250 °C. The column was maintained at 60 °C for 1 min, then

ramped at 30 °C/min to 300 °C with 3 min hold, and the total running time was 12 min. The FID

detector was used with an airflow of 400 mL/min, a H2 fuel flow of 25 mL/min and a N2 makeup flow

of 10 mL/min. The software used was Agilent OpenLAB CDS ChemStation Edition Rev.

C.01.09[144].

S5

2. Analytical methods

2.1. GC-FID analysis

Extraction procedure for GC-FID analysis

A 0.5 mL aliquot of the reaction mixture was added to 0.5 mL of EA, followed by vortexing for 30 s.

The EA layer was separated and dried over anhydrous Na2SO4. 40 µL of the dry EA layer was diluted

with 960 µL of EA and subjected to GC-FID analysis.

Yield determination and error analysis for GC-FID analysis

1 mL standard aqueous solutions of thioesters at different concentrations (1 mM, 2 mM, 5 mM, 10

mM, 20 mM, 40 mM) were prepared by dilution 100 mM stock solutions of these thioesters. 500 µL

of each standard solution were extracted using the above described extraction procedure for GC-FID

analysis. For each thioester, a six-point graph was plotted, correlating the characteristic GC peak (as

integrated automatically by the Agilent OpenLAB CDS ChemStation Edition Rev. C.01.09[144]

software) with substrate concentration (Figure S-1 – S-2). Each data point was obtained from three

independent measurements and the correlation line was obtained from the least-squares fitting

(intercept = 0). Error bars on graphs are shown as ± standard deviation for each data point. Overall

percentage error of the response factor corresponds to ± standard deviation for each slope value.

The concentrations of the compounds were calculated by comparing the product peak area with the

calibration curve. The yields of the products were calculated by multiplication of the determined

concentration with the reaction volume, divided by the amount of limiting starting material (in most

cases 0.1 mmol of the thio compound). Each reaction was performed at least three times to ensure

reproducibility and reported percentage yields are an average of these three runs, with an error

corresponding to ± standard deviation.

S6

Figure S-1: Correlation between the concentration of an aqueous solution of 3b and the measured gas

chromatography FID peak area.

Figure S-2: Correlation between the concentration of an aqueous solution of 3c and the measured gas

chromatography FID peak area.

R² = 0,9984

0

100

200

300

400

500

600

0 10 20 30 40 50

Pe

ak

are

a,

pA

*s

Concentration, mmol/L

R² = 0,9998

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

Pe

ak

are

a,

pA

*s


N,S-diacetyl cysteamine (3b)

Response factor: 11.773

S-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)

ethanethioate (3c)

Response factor: 31.655

S7

2.2. LCMS analysis

Sample preparation of LCMS analysis

After treatment of 1 mL of the reaction mixture with Chelex® 100 resin followed by centrifugation 20

µL of the supernatant were diluted with 180 µL of MilliQ-water and subjected to LCMS analysis

(injection volume = 0.1 µL unless otherwise noted).

Yield determination and error analysis for LCMS analysis

1 mL standard aqueous solutions of authentic samples at different concentrations (selection of 0.5

mM, 1 mM, 2 mM, 4 mM, 5 mM, 10 mM, 20 mM, 40 mM, 80 mM) were prepared by dilution of 100

mM stock solutions of these compounds. For each of these solutions 20 µL were diluted with 180 µL

MilliQ-water and subjected to LCMS-analysis. For each compound, a six-point graph was plotted,

correlating the characteristic LCMS-peak (as integrated by the Thermo Xcalibur 4.2.28.14 Qual

Browser software) with substrate concentration (Figure S-3 – S-7). Each data point was obtained from

three independent measurements and the correlation line was obtained from the least-squares fitting

(intercept = 0). Error bars on graphs are shown as ± standard deviation for each data point. Overall

percentage error of the response factor corresponds to ± standard deviation for each slope value.

The concentrations of the compounds were calculated by comparing the product peak area with the

calibration curve. The yields of the products were calculated by multiplication of the determined

concentration with the reaction volume, divided by the amount of limiting starting material (in most

cases 0.1 mmol of the thio compound). Each reaction was performed at least three times to ensure

reproducibility, and reported percentage yields are an average of these three runs, with an error

corresponding to ± standard deviation.

Figure S-3: Correlation between the concentration of an aqueous solution of 5b and the measured

LCMS peak area (m/z = 219.95–220.15).

y = 3,95597E+07x

R² = 0,9988

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

3,50E+09

0 20 40 60 80 100

Pe

ak

Are

a (

m/z

= 2

19

.95

-22

0.1

5)


4-[[2-(Acetylamino)ethyl]thio]-4-

oxobutanoic acid (5b)

Response factor: 3.95597*10^7

S8


LCMS peak area (m/z = 205.54–206.54).


LCMS peak area (m/z = 236.04–236.06).

y = 3,64713E+07x

R² = 0,9950

0,00E+00

2,00E+08

4,00E+08

6,00E+08

8,00E+08

1,00E+09

1,20E+09

1,40E+09

1,60E+09

0 10 20 30 40 50

Pe

ak

Are

a (

m/z

= 2

05

.54

-20

6.5

4)


y = 1,08917E+07x

R² = 0,9997

0,00E+00

5,00E+07

1,00E+08

1,50E+08

2,00E+08

2,50E+08

0 5 10 15 20 25

Pe

ak

Are

a (

23

6.0

4-2

36

.06

)


3-((2-acetamidoethyl)thio)-3-

oxopropanoic acid (8b)


4-((2-acetamidoethyl)thio)-2-hydroxy-4-

oxobutanoic acid (9b)


S9


LCMS peak area (m/z = 119.95–120.15).

Figure S-7: Correlation between the concentration of an aqueous solution of 1d and the measured

LCMS peak area (m/z = 236.97–237.17).

y = 2,55663E+07x

R² = 0,9973

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

0 20 40 60 80 100

Pe

ak

Are

a (

m/z

= 1

19

.95

-12

0.1

5)


y = 8,26212E+07x

R² = 0,9927

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

3,50E+09

4,00E+09

0 10 20 30 40 50

Pe

ak

Are

a (

m/z

= 2

36

.97

-23

7.1

7)

Concentration (mmol/L)

N,N'-(disulfanediylbis(ethane-2,1-

diyl))diacetamide (1d)


N -acetylcysteamine (1b)


S10


LCMS peak area (m/z = 161.95–161.15).

y = 5,72370E+07x

R² = 0,99995

0,00E+00

1,00E+09

2,00E+09

3,00E+09

4,00E+09

5,00E+09

0 20 40 60 80 100

Pe

ak

Are

a (

m/z

= 1

61

.95

-16

2.1

5)


N,S-diacetylcysteamine (3b)


S11

2.3. GC-MS analysis

Derivatisation procedure for GC-MS analysis

To facilitate GC-MS analysis, a literature ECF-derivatization procedure1,2

was applied to the sample to

convert carboxy groups to ethyl esters, hydroxy groups to ethyl carbonates, ketones to diethyl ketals,

and aldehydes to diethyl acetals, using a mixture of ethanol/ethyl chloroformate (EtOH/ECF). For

optimal gas chromatography resolution, heavy metal atoms were first removed using a resin (Chelex®

100 sodium form).

A ca. 1 mL aliquot of the reaction mixture was added to 50 mg of Chelex®, briefly shaken and then

centrifuged (6000 rpm, 3 min). To 600 µL of the supernatant was added EtOH (300 µL) and pyridine

(40 µL), followed by ethyl chloroformate (ECF, 40 µL). The mixture vortexed for 30 s. A second 40

µL portion of ECF was added and the mixture was vortexed again for 30 s. Next, CHCl3 (200 µL) was

added, followed by vortexing (10 s). Finally, NaHCO3 (600 µL) was added and the mixture was

vortexed again for 10 s. The CHCl3 layer was separated and dried over anhydrous Na2SO4. 50 µL of

the dry CHCl3 layer were diluted with 150 µL of ethyl acetate prior to GC-MS analysis.

Product identification

Reaction products derivatized were identified by comparing the mass spectra and retention times with

analogously derivatized authentic samples, as described in a previous paper.3

2.4. NMR analysis with water suppression

For 1H-NMR analysis using water suppression the samples were first treated with Chelex® 100 resin

followed by centrifugation to remove heavy metal ions. 600 µL from the supernatant were mixed with

100 µL of DSS in D2O (0.05 M) and analyzed by NMR with water suppression. Water suppression

was achieved using the Bruker ZGESGP pulse program. Relaxation delay D1 was set to 87 s, with

time domain size TD = 32768 and sweep width SWH = 4789.27 Hz (11.963 ppm). 32 scans were

acquired for each sample.

S12

3. Synthetic procedures

Compound Structure New Lit 1H

13C GC-

FID

HRMS

10

No 4 + - - -

1c

No 5 + + + -

1d

NH

3

4

S21

O

S

HN

O

No 6 + + + -

3b

No 7 + + + -

3c

No 8 + + + -

5b

No 9 + + - -

11 Yes - + + + +

8b

O

23

O

76S

4

5

NH

1HO

O

No 10

+ + - -

12

No 11

+ + - -

13

Yes - + + - +

9b

Yes - + + - +

S13

3.1. Synthesis of starting materials

Scheme S- 1: Synthesis routes toward 1d and 1c.

N,N'-(disulfanediylbis(ethane-2,1-diyl))diacetamide (1d)6

NH

3

4

S21

O

S

HN

O To a solution of N-acetylcysteamine 1b (1.00 g, 8.39 mmol,

1.0 equiv) in 9 mL of EA was added potassium iodide (7.0 mg, 0.042 mmol, 0.5 mol%) and aqueous

hydrogen peroxide (33 wt.%, 857 µL, 9.23 mmol, 1.1 equiv) subsequently and the mixture was stirred

at room temperature. After one hour the resulting brown solution was treated with Na2S2O3-solution (5

wt.%, 10 mL) followed by further stirring for 5 minutes. Then the layers were separated, and the

aqueous layer was extracted two more times with EA (2 x 20 mL). The combined organic layers were

dried (Na2SO4) and filtered. The solvent of the filtrate was removed in vacuo which gave access to 1d

(653 mg, 2.76 mmol, 66%) as a colorless white powder.

m.p. 87–88°C 1H-NMR (500 MHz, CDCl3): δ = 2.03 (s, 6 H, 1-H), 2.83 (t,

3J4,3 = 6.6 Hz, 4 H, 4-H),

3.56 (dt, 3J3,4 ≈

3J3,NH = 6.4 Hz, 4 H, 3-H), 6.68 (bs, 2 H, N-H).

13C-NMR (125 MHz, CDCl3): δ =

23.1, 37.7, 38.6, 170.9.

S14

2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (10)4

For the synthesis of the tosylate to a solution of

triethylene glycol monomethyl ether (12.3 g, 75 mmol, 1.0 equiv) in 150 mL DCM was added

triethylamine (11.5 mL, 82 mmol, 1.1 equiv), 4-dimethylaminopyridine (0.46 g, 3.75 mmol, 5 mol%)

and tosyl chloride (14.3 g, 75 mmol, 1.0 equiv). After stirring the reaction mixture for 5 hours at room

temperature DCM (100 mL) was added and the organic layer was washed with 1 M HCl solution (150

mL). The layers were separated, and the organic layer was dried (Na2SO4) and filtered. After removal

of the solvent in vacuo the crude tosylate 10 (21.9 g) was isolated as a yellow oil and used in the next

step without further purification.

1H-NMR (400 MHz, CDCl3): δ = 2.44 (s, 3 H, 12-H), 3.36 (s, 3 H, 1-H), 3.53 (m, 2 H, 2-H),

3.56–3.63 (m, 6 H, 3-H, 4-H, 5-H), 3.68 (t, 3J6,7 = 4.8 Hz, 2 H, 6-H), 4.15 (t,

3J7,6 = 4.8 Hz, 2

H, 7-H), 7.33 (d, 3J10,9 = 8.1 Hz, 2 H, 10-H), 7.79 (d,

3J9,10 = 8.2 Hz, 2 H, 9-H).

S-(2-(2-(2-methoxyethoxy)ethoxy)ethyl) ethanethioate (3c)8

To a solution of the tosylate 10 (21.9 g, 68.7 mmol, 1.0 equiv)

in 100 mL DMF under N2-atmosphere was added potassium iodide (0.57 g, 3.44 mmol, 5 mol%) and

potassium thioacetate (9.42 g, 82 mmol, 1.2 equiv). The mixture was stirred at room temperature

overnight. After 16 hours 300 mL of EA were added followed by washing with water (3 x 150 mL).

The organic layer was dried (Na2SO4) and filtered. After removal of the solvent in vacuo the thioester

3c (10.9 g, 49.2 mmol, 66% over two steps) was isolated as a yellow oil. The compound was used for

the synthesis of 1c and also as an authentic sample for the acetylation reaction.

1H-NMR (400 MHz, CDCl3): δ = 2.32 (s, 3 H, 9-H), 3.08 (t,

3J7,6 = 6.5 Hz, 2 H, 7-H), 3.37 (s, 3 H, 1-

H), 3.54 (m, 2 H, 2-H), 3.56–3.66 (m, 8 H, 3-H, 4-H, 5-H, 6-H). 13

C-NMR (100 MHz, CDCl3): δ =

28.8, 30.5, 59.0, 69.7, 70.3, 70.5, 70.5, 71.9, 195.5.

S15

2-(2-(2-methoxyethoxy)ethoxy)ethane-1-thiol (1c)5

For the synthesis of the thiol to a slurry of potassium

carbonate (9.33 g, 67.5 mmol, 1.5 equiv) in 45 mL of methanol was added a solution of 3c (10.0 g, 45

mmol, 1.0 equiv) dropwise over 30 seconds in the dark. After stirring at room temperature for one

hour the reaction mixture was diluted with DCM (300 mL) and washed with sat. NH4Cl solution. The

aqueous layer was extracted with DCM (100 mL) and the combined organic layers were dried

(Na2SO4). After filtration the solvent was removed under reduced pressure giving access to 1c (7.75 g,

43 mmol, 96%) as a pale yellow oil.

1H-NMR (400 MHz, CDCl3): δ = 1.57 (t,

3JSH,7 = 8.3 Hz, 1 H, S-H), 2.68 (dt,

3J7,SH = 8.3 Hz,

3J7,6 =

6.4 Hz, 2 H, 7-H), 3.37 (s, 3 H, 1-H), 3.50–3.68 (m, 10 H, 2-H, 3-H, 4-H, 5-H, 6-H). 13

C-NMR (100

MHz, CDCl3): δ = 24.3, 59.0, 70.2, 70.6, 70.6, 71.9, 72.9.

S16

3.2. Synthesis of reference compounds

Scheme S- 2: Synthesis routes toward several reference compounds.

S17

N,S-diacetyl cysteamine (3b)7

To a solution of N-acetyl cysteamine 1b (2.00 g, 16.8 mmol, 1.0 equiv) in 50

mL of DCM was added DIPEA (4.4 mL, 25.2 mmol, 1.5 equiv) followed by the dropwise addition of

acetyl chloride (1.2 mL, 17.6 mmol, 1.05 equiv). The reaction mixture was stirred at room

temperature. After 1.5 hours the mixture was diluted with DCM (50 mL) and washed with 1 M HCl

solution (30 mL) and sat. NaHCO3-solution (10 mL). The organic layer was dried (Na2SO4) and

filtered. Removal of the solvent under reduced pressure afforded 3b (2.00 g, 12.4 mmol, 74%) as a

pale yellow oil.

1H-NMR (300 MHz, CDCl3): δ = 1.95 (s, 3 H, 1-H), 2.34 (s, 3 H, 6-H), 3.00 (t,

3J4,3 = 6.5 Hz, 2 H, 4-

H), 3.40 (dt, 3J3,4 ≈

3J3,NH = 6.3 Hz, 2 H, 3-H), 6.03 (br. s, 1 H, N-H).

13C-NMR (75 MHz, CDCl3): δ =

23.1, 28.7, 30.6, 39.5, 170.4, 196.3.

4-[[2-(Acetylamino)ethyl]thio]-4-oxobutanoic acid (5b)9

To a solution of N-acetyl cysteamine 1b (99.2 mg, 0.83 mmol,

1.0 equiv) in 3 mL of acetonitrile was added DMAP (10.2 mg, 0.083 mmol, 0.1 equiv), pyridine

(197.4 mg, 2.50 mmol, 3.0 equiv) and succinic anhydride (83.26 mg, 0.83 mmol, 1.0 equiv). The

reaction mixture was stirred at room temperature. After 2 hours the mixture was diluted with sat.

NaHCO3-solution (3 mL) and washed with Et2O (5 mL). The organic layer was discarded. The

aqueous layer was acidified with 1 M HCl solution to pH 1 and was extracted with EA (4 × 10 mL).

The combined organic layers were dried (Na2SO4). After filtration the solvent was removed under

reduced pressure giving access to 5b (88 mg, 0.40 mmol, 48 %) as a colorless powder.

m.p. 78–80 °C 1H-NMR (500 MHz, CDCl3): δ = 1.97 (s, 3 H, 8-H), 2.73 (t,

3J3,2 = 6.6 Hz, 2 H, 3-H),

2.90 (t, 3J2,3 = 6.6 Hz, 2 H, 2-H), 3.06 (t,

3J5,6 = 6.2 Hz, 2 H, 5-H), 3.45 (dt,

3J6,5 =

3J6,N-H = 6.2 Hz, 2 H,

6-H), 5.99 (s, 1 H, N-H). 13

C-NMR (125 MHz, CDCl3): δ = 23.1, 28.5, 28.9, 38.3, 39.7, 171.0, 175.5,

198.5.

S18

3-((2-acetamidoethyl)thio)-3-oxopropanoic acid (8b)10

O

23

O

76S

4

5

NH

1HO

O To a solution of malonyl monothiophenylester12

(125 mg, 0.64 mmol,

1.0 equiv) and NaHCO3 (107 mg, 1.27 mmol, 2.0 equiv) in 1 mL of MilliQ water was added N-

acetylcysteamine 1b (85 mg, 0.71 mmol, 1.1 equiv) dissolved in 1 mL of MilliQ water over 30

minutes. After stirring at room temperature for one hour, the mixture was acidified to pH = 1 with 1 M

HCl. The aqueous layer was washed with chloroform (3 x 10 mL) and DCM (2 x 10 mL). The

aqueous layer was lyophilizd. The resulting solid was suspended in acetonitrile (20 mL) and filtered to

remove NaCl. The filtrate was dried in vacuo giving access to the malonyl thioester 8b (80 mg, 0.39

mmol, 61 %) as a pale yellow oil.

1H-NMR (500 MHz, CDCl3, DMSO-d6): δ = 1.81 (s, 3 H, 7-H), 2.95 (t,

3J4,5 = 6.6 Hz, 2 H, 4-H), 3.27

(dt, 3J5,4 ≈

3J5,NH = 6.4 Hz, 2 H, 5-H), 3.43 (s, 2 H, 2-H), 6.76 (br. s, 1 H, N-H).

13C-NMR (125 MHz,

CDCl3, DMSO-d6): δ = 22.7, 28.7, 38.6, 49.3, 167.6, 170.3, 191.5.

2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (12)11

A solution of racemic malic acid (3.00 g, 22.4 mmol, 1.0 equiv) and p-toluene

sulfonic acid monohydrate (85 mg, 0.45 mmol, 2 mol%) in a mixture of 25 mL of 2,2-

dimethoxypropane and 25 mL acetone was stirred at room temperature for 5 hours. Then sodium

bicarbonate (38 mg, 0.45 mmol, 2 mol%) and 50 mL of water were added. The mixture was extracted

with DCM (3 x 100 mL) and the combined organic layers were dried (Na2SO4). After filtration and

removal of the solvent in vacuo the crude product was recrystallized from PE/Et2O (97:3). The

protected malic acid 12 (1.74 g, 10.0 mmol, 45%) was isolated as a white solid.

m.p. 74–77°C 1

H-NMR (400 MHz, CDCl3): δ = 1.56 (s, 3 H, 6-H), 1.61 (s, 3 H, 6-H'), 2.85 (dd,

2J2a,2b = 17.2 Hz,

3J2a,3 = 6.6 Hz, 1 H, 2-Ha), 2.99 (dd,

2J2b,2a = 17.2 Hz,

3J2b,3 = 3.8 Hz, 1 H, 2-

Hb), 4.71 (dd, 3J3,2a = 6.6 Hz,

3J3,2b = 3.8 Hz, 1 H, 3-H), 10.23 (br. s, 1 H, COO-H). 13

C-NMR

(100 MHz, CDCl3): δ = 25.8, 26.7, 36.0, 70.4, 111.4, 171.8, 175.1.

S19

S-(2-acetamidoethyl) 2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)ethanethioate (13)

To a mixture of the protected malic acid 12 (300 mg, 1.72 mmol, 1.05

equiv) and DIPEA (344 µL, 1.97 mmol, 1.2 equiv) in 17 mL of DCM was added ethyl chloroformate

(158 µL, 1.64 mmol, 1.0 equiv) dropwise at 0 °C. After 5 minutes, N-acetylcysteamine 1b (196 mg,

1.64 mmol, 1.0 equiv) was added and the mixture was allowed to warm to room temperature. After 3

hours the reaction mixture was diluted with DCM (30 mL) and washed once with 1 M HCl-solution

(10 mL) and sat. NaHCO3-solution (10 mL). The organic layer was dried (Na2SO4) and filtered. After

removal of the solvent in vacuo the thioester 13 (369 mg, 1.34 mmol, 82%) was isolated as a colorless

liquid.

1H-NMR (500 MHz, CDCl3): δ = 1.57 (s, 3 H, 10-H), 1.62 (s, 3 H, 10-H'), 2.00 (s, 3 H, 8-H),

3.06 (dd, 2J3a,3b = 16.5 Hz,

3J3a,2 = 6.7 Hz, 1 H, 3-Ha), 3.11 (td,

3J5,6 = 6.4 Hz,

4J5,NH = 2.1 Hz,

2 H, 5-H), 3.19 (dd, 2J3b,3a = 16.5 Hz,

3J3b,2 = 4.0 Hz, 1 H, 3-Hb), 3.46 (m, 2 H, 6-H), 4.76 (dd,

3J2,3a = 6.7 Hz,

3J2,3b = 4.3 Hz, 1 H, 2-H), 6.29 (br. s, 1 H, N-H). 13

C-NMR (100 MHz, CDCl3): δ

= 23.0, 25.8, 26.8, 28.8, 39.3, 44.8, 70.5, 111.4, 170.7, 171.8, 195.0. HRMS: [C11H17NO5S+K]*

calculated 314.0450 found: 314.0459, Rf(silica, EA) = 0.40.

4-((2-acetamidoethyl)thio)-2-hydroxy-4-oxobutanoic acid (9b)

A solution of the thioester 13 (87 mg, 0.317 mmol, 1.0 equiv) in a

mixture of 2.5 mL THF and 1 mL of 1 M HCl solution was heated to 50 °C. After one hour, TLC-

analysis showed complete conversion of the starting material. The reaction was cooled to room

temperature and extracted with EA (5 mL). The organic layer was again extracted with 2 mL of 1 M

HCl-solution and the combined aqueous layers were dried in vacuo giving access to the malyl

thioester 9b (51.4 mg, 0.218 mmol, 69%) as a colorless resin.

S20

1H-NMR (500 MHz, D2O): δ = 1.89 (s, 3 H, 8-H), 3.01 (m, 2 H, 5-H), 3.07 (dd,

2J3a,3b = 16. 2 Hz,

3J3a,2

= 6.7 Hz, 1 H, 3-Ha), 3.13 (dd, 2J3b,3a = 15.9 Hz,

3J3b,2 = 4.6 Hz, 1 H, 3-Hb), 3.32 (t,

3J6,5 = 6.3 Hz, 2 H,

6-H), 4.57 (dd, 3J2,3a = 6.9 Hz,

3J2,3b = 4.1 Hz, 1 H, 2-H).

13C-NMR (125 MHz, DMSO-d6): δ = 25.2,

30.8, 40.8, 50.6, 69.6, 172.0, 176.8, 198.4. HRMS: [C8H13NO5S+H]* calculated 236.0587 found:

236.0583.

2,5,8,15,18,21-hexaoxa-11,12-dithiadocosane (11)

A solution of the thiol 1c (300 mg,

1.66 mmol, 1.0 equiv) in 2 mL of EA was treated with sodium iodide (1 mg, 8 µmol, 0.5 mol%) and

aqueous hydrogen peroxide solution (33 wt.%, 160 µL, 1.83 mmol, 1.1 equiv) and stirred at room

temperature. After one hour the resulting brown solution was treated with Na2S2O3 solution (5 wt.%, 2

mL), followed by further stirring for 5 minutes. Then the layers were separated, and the aqueous layer

was extracted two more times with EA (2 x 5 mL). The combined organic layers were dried (Na2SO4)

and filtered. The solvent of the filtrate was removed in vacuo which gave access to 11 (287 mg, 0.80

mmol, 96%) as a pale yellow oil.

1H-NMR (400 MHz, CDCl3): δ = 2.88 (t,

3J7,6 = 6.7 Hz, 4 H, 7-H), 3.37 (s, 6 H, 1-H), 3.55 (m, 4 H, 2-

H), 3.62–3.72 (m, 12 H, 3-H, 4-H, 5-H), 3.72 (t, 3J6,7 = 6.7 Hz, 4 H, 6-H).

13C-NMR (125 MHz,

CDCl3): δ = 38.3, 59.0, 69.6, 70.3, 70.51, 71.52, 71.9. HRMS: [C14H30O6S2+Na]+ calculated:

381.1376 found: 381.1384.

S21

3.3. Formation of thioesters under prebiotic conditions

General procedures for thioester formation:

Method A (K2S2O8, FeS, 70 °C):

To a solution of the thiol (0.1 mmol) in 3 mL of MilliQ water in a Pyrex pressure tube with a stirring

bar was added the corresponding α-keto acid (0.50 mmol; 0.42 mmol for hydroxyketoglutarate) or

acetaldehyde (1.0 mmol) under a constant argon flow. Iron(II) sulfide (0.05 mmol) and potassium

persulfate (0.2 mmol) were added. The reaction vessel was closed and heated to 70 °C for 3 hours.

After cooling to room temperature, the amount of thioester formed was determined by GC-FID

analysis or LCMS analysis (see section 2.1 and 2.2). Additionally selected samples were analyzed by

NMR using water suppression (see section 2.4).

Method B (K2S2O8, FeS, UV-A):

To a solution of the thiol (0.1 mmol) in 3 mL of MilliQ water in a quartz tube with a stirring bar was

added the corresponding α-keto acid (0.50 mmol; 0.42 mmol for hydroxyketoglutarate) or

acetaldehyde (1.0 mmol) under a constant argon flow. Iron(II) sulfide (0.05 mmol) and potassium

persulfate (0.2 mmol) were added. The tube was closed with a septum and irradiated in a Luzchem

LZC-ORG photoreactor equipped with 10 LZC-UVA lamps for 6 hours. The amount of thioester

formed was determined by GC-FID analysis or LCMS analysis (see section 2.1 and 2.2). Additionally

selected samples were analyzed by NMR using water suppression (see section 2.4).

Method C (KHSO4, UV-A):

To a solution of the thiol (0.1 mmol) in 3 mL of a KHSO4-solution (3 M in MilliQ water) in a quartz

tube with a stirring bar was added the corresponding α-keto acid (0.50 mmol; 0.42 mmol for

hydroxyketoglutarate) or acetaldehyde (1.0 mmol) under a constant argon flow. The tube was closed

with a septum and irradiated in a Luzchem LZC-ORG photoreactor equipped with 10 LZC-UVA

lamps for 6 hours. The amount of thioester formed was determined by GC-FID analysis or LCMS

analysis (see section 2.1 and 2.2). Additionally selected samples were analyzed by NMR using water

suppression (see section 2.4).

S22

Hydroxyketoglutarate was synthesized by adapting a literature procedure without isolation of the

product.3 To a solution of glyoxylic acid hydrate (506 mg, 5.50 mmol, 1.1 equiv) in 30 mL of MilliQ

water was added oxaloacetic acid (660 mg, 5.00 mmol, 1.0 equiv) and NaHCO3 (840 mg, 10.0 mmol,

2.0 equiv) under argon. The resulting mixture was stirred at room temperature for two hours. A

concentration of 139 mM for the resulting hydroxyketoglutarate was determined by NMR analysis

with water suppression using DSS as an internal standard.

S23

Table S-1: Optimization of the oxidative decarboxylation of sodium pyruvate with 1b.

Entry Additive (equiv/conc) Light source/T t [h] Yield(3b) [%]

1 none UVA/-B/-C/CW 3 0.6 ± 0.2

2 none 70 °C 3 0.0 ± 0.0

3 K2S2O8 (2 equiv) rt 3 1.5 ± 0.7

4 K2S2O8 (2 equiv) 50 °C 3 9.0 ± 0.9

5 K2S2O8 (2 equiv) 70 °C 3 14.8 ± 0.7

6 K2S2O8 (2 equiv) 100 °C 3 8.6 ± 0.8

7 K2S2O8 (2 equiv) UVA/-B/-C/CW 3 6.4 ± 1.8

8 K2S2O8 (2 equiv) + FeS (0.5 equiv) UVA/-B/-C/CW 3 23.1 ± 2.7

9 K2S2O8 (2 equiv) + FeS (0.5 equiv) 70 °C 3 21.2 ± 0.5

10 KHSO4 (3 M) UVA 6 22.1 ± 0.4

11 Fe(ClO4)3 (1.0 equiv) UVA 6 4.8 ± 0.4

12 K2S2O8 (2 equiv) + FeS (0.5 equiv) UVA 6 31.2 ± 0.4

13 K2S2O8 (2 equiv) + FeS (0.5 equiv) 70 °C 3 26.6 ± 0.7

14 KHSO4 (1 M) UVA/-B/-C/CW 3 6.2 ± 0.2

15 KHSO4 (3 M) UVA/-B/-C/CW 3 8.4 ± 1.9

16 NaHSO4 (1 M) UVA/-B/-C/CW 3 4.4 ± 0.9

17 H2SO4 (1 M) UVA/-B/-C/CW 3 6.8 ± 0.3

18 H3PO4 (3 M) UVA/-B/-C/CW 3 6.1 ± 0.3

19 HCl (0.5 M) UVA/-B/-C/CW 3 2.5 ± 0.2

20 H2SO4 (1 M) or H3PO4 (3 M) or HCl

(0.5 M) or KHSO4 (3 M) 70 °C 3 0 ± 0

21 nonea UVA/-B/-C/CW 3 1.8 ± 0.0

22 KHSO4 (1 M) none 3 0.5 ± 0.1

23 KHSO4 (1 M) UVA 3 14.2 ± 0.7

24 KHSO4 (1 M) UVB 3 9.5 ± 1.0

25 KHSO4 (1 M) UVC 3 0.8 ± 0.0

26 KHSO4 (1 M) CW 3 0.6 ± 0.1

27 KHSO4 (3 M) b UVA 6 4.9 ± 0.2

28 Fe(ClO4)3 (1.0 equiv) b

UVA 6 0.5 ± 0.1

29 K2S2O8 (2 equiv) + FeS (0.5 equiv) b UVA 6 31.9 ± 0.7

S24

Entry Additive (equiv/conc) Light source/T t [h] Yield(3b) [%]

30 K2S2O8 (2 equiv) + FeS (0.5 equiv) b 70 °C 3 32.9 ± 0.8

31 KHSO4 (0.5 M) UVA 3 7.2 ± 0.5

32 KHSO4 (0.41 M) + K2SO4 (0.09 M) UVA 3 6.6 ± 0.3

33 KHSO4 (0.33 M) + K2SO4 (0.17 M) UVA 3 5.5 ± 0.3

34 KHSO4 (0.25 M) + K2SO4 (0.25 M) UVA 3 4.4 ± 0.7

35 KHSO4 (0.17 M) + K2SO4 (0.33 M) UVA 3 3.0 ± 0.1

36 KHSO4 (0.09 M) + K2SO4 (0.41 M) UVA 3 1.4 ± 0.1

37 KHSO4 (0.46 M) + K2SO4 (0.04 M) UVA 3 0.8 ± 0.1

38 K2SO4 (0.5 M) UVA 3 0.5 ± 0.0

39 Fe(ClO4)3 (2 equiv) UVA/-B/-C/CW 3 4.4 ± 0.3

40 FeCl2 (2 equiv) UVA/-B/-C/CW 3 2.8 ± 0.1

41 FeCl3 (2 equiv) UVA/-B/-C/CW 3 3.1 ± 0.1

42 FeS (2 equiv) UVA/-B/-C/CW 3 1.1 ± 0.2

43 KHSO4 (3 M)c UVA 6 0.0 ± 0.0

44 K2S2O8 (2 equiv) + FeS (0.5 equiv)c UVA 6 0.0 ± 0.0

45 K2S2O8 (2 equiv) + FeS (0.5 equiv)c 70 °C 3 0.0 ± 0.0

46 KHSO4 (3 M)d UVA 6 0.0 ± 0.0

47 K2S2O8 (2 equiv) + FeS (0.5 equiv)d UVA 6 0.0 ± 0.0

48 K2S2O8 (2 equiv) + FeS (0.5 equiv)d 70 °C 3 0.0 ± 0.0

a pyruvic acid was used instead of sodium pyruvate

b 0.5 equiv disulfide 1d instead of thiol 1b.

ccontrol experiment without sodium pyruvate

dcontrol experiment without thiol 1b

To show that the described method can also be applied to other thiols, the formation of the acetyl

thioester 3c starting from pyruvate and the thiol 1c has been investigated.

Scheme S-3: Thioester formation with an alternative thiol 1c analyzed by GC-FID.

S25

Additionally, the use of α-hydroxy acids instead of α-keto acids has been investigated.

Scheme S-4: Thioester formation using lactic acid analyzed by GC-FID.

Figure S-9: Time dependence of the thioester formation under optimized conditions

0

10

20

30

40

50

60

0 10 20 30 40 50

yie

ld 3

b[%

]

time [h]

K2S2O8 + FeS, 70°C

K2S2O8 + FeS, UVA

KHSO4, UVA

S26

Table S-2: Stability and decomposition products of different keto acids using methods A–C

Me

O

O-

OMe S

O

R

pyruvate (2)

O

O-

O

oxaloacetate 6 (n = 1)

α-ketoglutarate 4 (n = 2)

HO

O

n S

O

HO

O

n

R

malonyl thioester 8b (n = 1)

succinyl thioester 5b (n = 2)

acetyl thioester (3b/3c)RSH (1b/1c)

or RSSR (1d)

H2O

Ketoacid Thio-cmpd. Method Remaining Ketoacida

Decompositiona

pyruvate 1b A 48%b acetate (7%)

pyruvate 1b B 48%b acetate (10%)

pyruvate 1b C 64%b acetate (2%)

pyruvate 1c A 63%b acetate (4%)

pyruvate 1c B 48%b acetate (9%)

pyruvate 1c C 63%b acetate (4%)

pyruvate 1d A 54%b acetate (4%)

pyruvate 1d B 56%b acetate (13%)

pyruvate 1d C 80%b acetate (1%)

α-ketoglutarate 1b A 66%b succinate (5%)

α-ketoglutarate 1b B 82% b succinate (7%)

α-ketoglutarate 1b C 57% b succinate (1%)

oxaloacetate 1b A 0% pyruvate (52%b)

oxaloacetate 1b B 0% pyruvate (32%b)

oxaloacetate 1b C 44%b pyruvate (4%

b)

a determined by NMR with DSS as internal standard using water suppression

b the shown value is the

sum of the ketoacid and its hydrate form

S27

3.4 Thioester formation from a non-enzymatic reaction network

A solution of sodium pyruvate 2 (110 mg, 1.0 mmol, 2.0 equiv), glyoxylic acid monohydrate (46 mg,

0.5 mmol, 1.0 equiv) and FeCl2∙4H2O (200 mg, 1.0 mmol, 2.0 equiv) in 3 mL of MilliQ water was

prepared in a Pyrex pressure tube equipped with a stirring bar under a constant argon flow. For

oxidation procedure A N-acetylcysteamine 1b (59 mg, 0.5 mmol, 1.0 equiv) was added. The reaction

vessel was closed and heated to 70 °C for 1 h or 3 h, followed by an oxidation procedure.

Oxidation procedure A:

To the solution prepared as described above, potassium persulfate (270 mg, 1.0 mmol, 1.0 equiv) was

added under a constant argon flow. The reaction vessel was closed and heated to 70 °C for 3 hours.

After cooling to room temperature, the amount of thioesters formed was determined by GC-FID

analysis or LCMS analysis (see section 2.1 and 2.2), the different intermediates of the network were

determined by GC-MS analysis (see section 2.3).

Oxidation procedure B: (in this case, N-acetylcysteamine was added at a later stage)

To the solution prepared as described above, KHSO4 (1.2 g, 9.0 mmol, to form a ~3 M solution) and

N-acetylcysteamine (59 mg, 0.50 mmol 1.0 equiv) were added, the mixture was transferred in a quartz

tube under a constant argon flow. The tube was closed with a septum and irradiated in a Luzchem

LZC-ORG photoreactor equipped with 10 LZC-UVA lamps for 3 hours. The amount of thioesters

formed was determined by GC-FID analysis or LCMS analysis (see section 2.1 and 2.2), the different

intermediates of the network were determined by GC-MS analysis (see section 2.3).

S28

Table S-3: Thioester formation from a non-enzymatic reaction network

Me

O

O

O

H

O

O

O

pyruvate

glyoxylate

FeCl2 (2 equiv)addition 1

conditions 1H2O

-O

O

O

-O

O

O

O-

O

α2εetoglutarate

hydroxyketoglutarate

Me

O

O

Opyruvate

Reaction networkwith 16 biological

intermediates

SR

O

-O

O

Me SR

O

CO2

O-

O

SR

O

-O

O

acetyl thioester (3b)

malyl thioester (9b)

succinyl thioester (5b)

OH

OH

addition 2conditions 2

Entry equiv pyruvate addition 1 (equiv) conditions 1 addition 2 (equiv) conditions 2 c(3b)a [mM] c(5b)

b [mM] c(9b)

b [mM]

1 2 70 °C, 1 h Thiol (1)

KHSO4 (3 M) UVA, 3 h 0.11 ± 0.01 0.19 ± 0.04 0.06 ± 0.00

2 2 Thiol (1) 70 °C, 1 h K2S2O8 (2) 70 °C, 3 h 1.41 ± 0.11 0.11 ± 0.01 3.60 ± 0.30

3 2 Thiol (1) 70 °C, 3 h K2S2O8 (2) 70 °C, 3 h 1.11 ± 0.14 0.11 ± 0.02 3.63 ± 0.28

4 1 Thiol (1)

KHSO4 (3 M) UVA, 3 h - - 0.06 ± 0.05 - -

5 2 Thiol (1)

KHSO4 (3 M) UVA, 6 h - - 0.16 ± 0.02 - -

6 1 Thiol (1)

KHSO4 (3 M) UVA, 3 h - - 0.11 ± 0.05 - -

7 2 Thiol (1)

KHSO4 (3 M) UVA, 6 h - - 0.31 ± 0.05 - -

8 2 Thiol (1) 70 °C, 1 h KHSO4 (3M) UVA, 3 h 0.12 ± 0.01 - -

9 2 Thiol (1) 70 °C, 1 h KHSO4 (3M) UVA, 6 h 0.15 ± 0.01 - -

10 2 Thiol (1) UVA, 1 h KHSO4 (3M) UVA, 3 h 0.22 ± 0.02

a Determined by GC-FID analysis after extraction with ethyl acetate

b Determined by LCMS-analysis. For the network experiments 0.5 µL instead of 0.1 µL were

injected, the resulting value calculated using the calibration curves was divided by 5 afterwards. See section 4.10 for GCMS-chromatograms of the network

products.

S29

4. Experimental data

4.1. NMR spectra of the synthesized compounds

N,N'-(disulfanediylbis(ethane-2,1-diyl))diacetamide (1d)

10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

6.1

2

4.0

0

4.0

9

1.7

76

.68

3.5

83

.56

3.5

53

.54

2.8

42

.83

2.8

1

2.0

3


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

17

0.9

3

38

.58

37

.69

23

.11

S30

2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (10)

8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

3.0

7

3.1

2

2.0

9

6.2

4

2.5

8

2.0

2

2.0

0

2.0

07

.80

7.7

8

7.3

47

.32

4.1

64

.15

4.1

43

.69

3.6

83

.67

3.3

6

2.4

4

S31

S-(2-(2-(2-methoxyethoxy)ethoxy)ethyl) ethanethioate (3c)


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

3.0

0

2.0

3

2.9

2

2.0

0

8.0

5

3.3

73

.09

3.0

83

.06

2.3

2


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

19

5.4

6

71

.90

70

.54

70

.51

70

.28

69

.72

59

.00

30

.50

28

.80

S32

2-(2-(2-methoxyethoxy)ethoxy)ethane-1-thiol (1c)


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

0.8

8

1.9

6

3.0

0

2.0

6

8.2

2

3.3

7

2.7

12

.69

2.6

72

.66

1.5

91

.57

1.5

5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

72

.88

71

.93

70

.56

70

.22

59

.03

24

.25

S33



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

2.8

9

2.9

9

2.0

2

2.0

0

0.8

35

.96

3.4

43

.43

3.4

13

.39

3.0

33

.01

3.0

0

2.3

4

1.9

6

240 220 200 180 160 140 120 100 80 60 40 20 0 -20Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

19

6.3

3

17

0.3

6

39

.52

30

.57

28

.71

23

.12

S34



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

4.0

0

5.9

44

.08

12

.30

4.0

1

3.7

43

.72

3.7

13

.65

3.6

43

.64

3.6

33

.56

3.5

43

.37

2.9

02

.88

2.8

6

180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

71.8

770.5

270.3

269.5

7

59.0

0

38.3

2

70.5 70.4 70.3 70.2Chemical Shift (ppm)

0.25

0.50

0.75

1.00

Norm

aliz

ed I

nte

nsity

70.5

270.5

1

70.3

2

S35

4-[[2-(Acetylamino)ethyl]thio]-4-oxobutanoic acid (5b)

9 8 7 6 5 4 3 2 1 0 -1Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

3.0

0

2.0

4

2.1

4

2.0

8

2.1

5

1.2

35.9

9

3.4

53.4

43.4

33.0

73.0

63.0

52.9

22.9

02.8

92.7

42.7

3

1.9

7

220 200 180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

Norm

aliz

ed I

nte

nsity

198.4

5

175.4

6170.9

9

39.6

538.2

4

28.8

928.4

6

23.0

4

S36

3-((2-acetamidoethyl)thio)-3-oxopropanoic acid (8b)

12 11 10 9 8 7 6 5 4 3 2 1 0 -1Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

3.0

3

2.0

0

2.1

4

1.9

6

1.0

36.7

6

3.4

33.2

93.2

83.2

63.2

52.9

62.9

52.9

3

1.8

1


0.05

0.10

0.15

0.20

0.25

0.30

Norm

aliz

ed I

nte

nsity

DMSO-d6

CDCl3

191.5

2

170.3

1167.6

1

49.2

7

38.6

1

28.7

0

22.6

7

S37

2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (12)

12 11 10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

3.0

5

2.9

7

1.0

2

1.0

0

0.9

5

0.9

610.2

3

4.7

24.7

14.7

04.7

0

3.0

13.0

12.9

82.9

72.8

82.8

62.8

4

1.6

11.5

6


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

175.1

3171.8

4

111.3

7

70.3

5

35.9

6

26.7

025.7

9

S38

S-(2-acetamidoethyl) 2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)ethanethioate (13)

10 9 8 7 6 5 4 3 2 1 0 -1Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

3.4

9

3.3

4

3.0

0

1.2

1

1.9

4

1.0

2

2.1

8

1.1

0

1.0

26.2

9

4.7

74.7

64.7

64.7

5

3.4

73.4

63.4

53.1

73.1

13.1

13.0

93.0

83.0

7

2.0

01.6

21.5

7

220 200 180 160 140 120 100 80 60 40 20 0 -20Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

195.0

0

171.8

0170.6

7

111.3

9

70.4

7

44.8

1

39.2

8

28.7

726.7

925.8

023.0

0

S39

4-((2-acetamidoethyl)thio)-2-hydroxy-4-oxobutanoic acid (9b)

9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed I

nte

nsity

3.0

0

2.2

6

2.0

4

2.0

6

1.0

44.5

84.5

74.5

74.5

6

3.3

33.3

23.3

03.1

23.1

13.0

93.0

83.0

23.0

2

1.8

9


0.05

0.10

0.15

0.20

No

rma

lize

d In

ten

sity

19

8.4

4

17

6.7

9

17

1.9

9

69

.55

50

.64

40

.83

30

.80

25

.19

S40

4.2. NMR spectra of reference compds. in H2O/D2O with DSS as IS with water suppression

N-acetyl cysteamine (1b) in H2O/D2O+DSS

8 7 6 5 4 3 2 1 0 -1Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

No

rma

lize

d In

ten

sity

3.0

0

1.8

0

1.5

5

0.4

7DSS DSSDSS DSS

3.3

93

.37

3.3

53

.33

2.9

32

.91

2.8

82

.85

2.6

82

.66

2.6

42

.00

1.8

11

.78

1.7

61

.73

0.6

50

.62

0.6

0

0.0

0

2-(2-(2-methoxyethoxy)ethoxy)ethane-1-thiol (1c) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

0.6

3

2.4

6

3.0

0

7.9

6

DSS DSS DSS DSS

3.6

9

3.3

72

.93

2.9

12

.88

2.7

3

1.7

5

0.6

50

.62

0.6

0

0.0

0

S41

N,N'-(disulfanediylbis(ethane-2,1-diyl))diacetamide (1d) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

6.0

0

4.1

3

2.9

2

DSS DSSDSS DSS

8.1

2

3.5

33

.51

3.4

93

.47

2.8

72

.85

2.8

31

.99

1.8

11

.78

1.7

71

.76

1.7

41

.73

1.7

00

.65

0.6

20

.60

0.0

0

N,S-diacetyl cysteamine (3b) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

2.9

4

3.0

0

2.2

3

1.8

4

0.4

3

DSS DSSDSS DSS

8.1

0

3.4

03

.38

3.3

63

.34

3.0

53

.03

3.0

12

.93

2.9

12

.88

2.3

71

.95

1.7

81

.75

1.7

41

.73

0.6

50

.62

0.6

0

0.0

0

S42

S-(2-(2-(2-methoxyethoxy)ethoxy)ethyl) ethanethioate (3c) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

3.0

0

2.0

3

2.3

2

6.3

5

DSSDSS DSSDSS

3.3

73

.14

3.1

23

.10

2.9

32

.90

2.8

82

.85

2.3

81

.81

1.7

91

.77

1.7

51

.74

1.7

21

.72

0.6

50

.62

0.6

0

0.0

0

4-[[2-(Acetylamino)ethyl]thio]-4-oxobutanoic acid (5b) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

3.0

0

2.6

3

5.5

6

2.0

4

2.0

1

DSS

5b+DSS

DSS

DSS

3.4

03

.38

3.3

63

.34

3.0

83

.06

3.0

42

.72

2.6

92

.67

1.9

5

S43

2,5,8,15,18,21-hexaoxa-11,12-dithiadocosane (11) in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

5.1

7

5.9

9

3.1

5

9.7

4

2.3

3DSSDSS

DSS

3.8

43

.82

3.8

0

3.3

7

2.9

72

.95

2.9

3

3-((2-acetamidoethyl)thio)-3-oxopropanoic acid (8b) in H2O/D2O+DSS

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)

0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

3.0

0

2.0

5

1.8

3

DSS

DSS

DSSDSS

3.4

33

.41

3.3

93

.37

3.1

23

.10

3.0

8

1.9

5

O

23

O

76S

4

5

NH

1HO

O

S44

sodium pyruvate (1)


0

0.25

0.50

No

rma

lize

d In

ten

sity

0.0

8

0.7

4

pyruvate-hydratepyruvate

2.3

7

1.4

8

0.0

0


0

0.25

0.50

No

rma

lize

d In

ten

sity

6.1

2

3.0

0

pyruvate-hydratepyruvate

2.4

7

1.5

9

0.0

010 9 8 7 6 5 4 3 2 1 0 -1

Chemical Shift (ppm)

0

0.25

0.50

No

rma

lize

d In

ten

sity

5.6

0

3.0

0

pyruvate pyruvate-hydrate

2.4

9

1.6

0

0.0

0

in H2O / D2O + DSS

in KHSO4 (1 M) / D2O + DSS


S45

oxaloacetic acid (6)


0

0.25

0.50

Norm

aliz

ed I

nte

nsity

2.0

03.0

2

0.0

0


0

0.25

0.50

Norm

aliz

ed I

nte

nsity

2.0

03.0

4

0.0

0


0

0.25

0.50

Norm

aliz

ed I

nte

nsity

2.0

03.0

7

0.0

0

in H2O / D2O + DSS



S46

α-ketoglutaric acid (4) in H2O/D2O+DSS


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

1.3

7

1.4

1

2.0

0

1.2

1

a-KG-hydrate

a-KG

a-KG-hydrate

a-KG

3.0

32

.75

2.7

22

.70

2.5

22

.49

2.4

72

.19

2.1

62

.14

succinic acid in H2O/D2O+DSS

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

4.0

02

.66

0.0

0

S47

malonic acid in H2O/D2O+DSS


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

2.0

03

.50

0.0

0

S48

4.3. NMR spectra of selected reaction mixtures showing prebiotic thioester formations

Table 1, Entry 1, Method A


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

7.9

0

9.7

9

3.0

0

6.1

3

3.5

8

18

.65

3b

1d

pyruvate, 3b

acetic acid

DSS

2.3

92

.38

2.0

91

.99

1.9

61

.62

Table 1, Entry 1, Method B


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

7.8

3

9.5

1

3.0

0

5.0

5

5.3

42

.97

15

.52

pyruvate

3b 1d

3b

pyruvate-hydrate

acetic acid

DSS

2.3

92

.37

2.0

91

.99

1.9

61

.60

S49

Table 1, Entry 1, Method C


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

18

.31

51

.96

3.1

4

19

.95

2.7

9

2.9

7

26

.10

acetic acid

3b 3b pyruvate-hydrate

1d

pyruvate

DSS

2.4

92

.39

2.1

12

.06

2.0

21

.60

0.0

0



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

7.8

8

8.6

7

3.0

0

6.7

4

2.3

8

19

.81

3b

1d pyruvate-hydrate

pyruvate, 3b

acetic acid

DSS

2.0

92

.00

1.9

61

.63

S50



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

6.5

3

9.3

8

3.0

0

6.4

5

5.6

8

2.9

6

15

.14

3b 3b

1d

pyruvate-hydrate

acetic acid

DSS

2.3

92

.37

2.0

91

.99

1.9

61

.58

0.0

0



0

0.1

0.2

0.3

0.4

0.5

No

rma

lize

d In

ten

sity

11

2.5

8

39

4.6

1

3.1

6

16

0.0

9

7.8

9

3.0

0

20

4.1

6

3b 3b

acetic acid

pyruvate-hydrate

1d

pyruvate

DSS

2.4

9

2.1

12

.06

2.0

2

1.6

0

0.0

0

S51


6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5 -1.0Chemical Shift (ppm)

0

0.25

0.50

0.75

1.00

Norm

aliz

ed I

nte

nsity

18.6

7

2.9

9

15.9

0

14.3

7

15.2

6

7.8

8

41.0

5

31.0

62.3

4

3.8

44.0

5

succinic acid 5b

1b, 1d

5b

a-KG

1d

a-KG-hydrate

a-KG-hydrate

1b, 5d

a-KG

DSS

3.5

13.4

93.3

83.3

63.0

83.0

63.0

42.9

92.7

42.7

12.6

92.6

72.5

32.5

12.4

82.2

42.2

22.1

91.9

91.9

5



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

8.0

9

3.0

0

6.8

7

5.5

8

6.2

0

4.7

1

26

.03

17

.19

2.6

4

2.7

2

1.0

7

succinic acid

5b

1b, 1d

5b

a-KG

a-KG-hydrate

a-KG

1d

1b, 5b

DSS

3.3

83

.36

3.0

63

.04

2.7

22

.70

2.6

72

.67

2.4

82

.46

2.1

92

.16

1.9

91

.99

1.9

5

0.0

0

S52



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

27

.67

3.0

0

22

.73

31

.06

35

.87

1.7

2

39

.45

20

.65

10

.59

7.6

9

succinic acid

5b

1b, 1d

a-KG

a-KG-hydrate

1d DSS

1b, 5b

5b, a-KG

3.5

43

.51

2.7

82

.75

2.7

32

.68

2.5

32

.51

2.4

82

.20

2.1

82

.15

2.0

31

.98

0.0

0



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

13

.53

26

.30

3.0

0

8.7

5

0.6

4

20

.80

2.8

9

3.6

2

pyruvate

1d3b

pyruvate-hydrate

8b

1d

1d, malonic acid

DSS

3.5

1

2.8

72

.85

2.8

3

2.4

22

.38

1.9

91

.96

1.5

9

0.0

0

S53


5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5 -1.0Chemical Shift (ppm)

0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

10

.58

10

.12

2.9

9

6.4

1

0.4

8

12

.71

2.3

3

2.2

8

3b

pyruvate

pyruvate-hydrate8b

1d

1d

1d, malonic acid

DSS

3.4

8

2.8

72

.85

2.8

3

2.4

02

.37

1.9

91

.95

1.6

1

0.0

0



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

9.0

0

2.3

0

5.9

61

7.8

5

2.6

1

pyruvate-hydrate

oxaloacetate

1d

DSS

1d, malonic acid

1d, 8b

3.0

7

S54



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

23

.10

44

.54

3.8

0

39

.05

3.0

1

6.2

8

2.8

9

13

.02

1d

3b

3b

acetaldehyde

DSS

3b acetaldehyde-hydrateacetaldehyde

9.6

99

.68

9.6

79

.66

8.1

3

3.4

03

.38

3.3

63

.34

2.8

72

.85

2.8

32

.38

2.2

42

.23

1.9

91

.96

1.3

31

.31



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d In

ten

sity

17

.99

3.0

0

3b

DSS2.3

9

S55



0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

21

.46

62

.95

3.1

5

14

.75

55

.54

3.0

1

17

.63

3b

1d

3b

acetaldehyde

acetaldehyde-hydrateacetaldehyde

9.6

79

.66

2.3

72

.24

2.2

32

.08

1.9

91

.96

1.3

31

.31

Scheme S-3 Method A


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

8.9

9

14

.56

2.4

6

23

.28

11

.55

acetic acid

pyruvate-hydrate

pyruvate, 3c

DSS

1c, 3c, disulfide-1c

3.3

8

2.3

9

2.0

9

1.6

1

0.0

0

S56

Scheme S-3 Method B


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

9.8

4

14

.78

6.0

9

3.0

0

16

.79

10

.20

3c

pyruvate


acetic acid

pyruvate-hydrate DSS

3.3

8

2.3

9

2.0

8

1.5

8

0.0

0

Scheme S-3 Method C


0

0.25

0.50

0.75

1.00

No

rma

lize

d In

ten

sity

18

.42

50

.89

3.5

0

3.0

1

26

.38

39

.00

acetic acid

pyruvate-hydrate

3c

pyruvate


DSS

2.1

1

S57

4.4. GC-FID chromatograms of authentic samples

N-acetylcysteamine (1b)

2-(2-(2-methoxyethoxy)ethoxy)ethane-1-thiol (1c)

S58

N,N'-(disulfanediylbis(ethane-2,1-diyl))diacetamide (1d)


S59

S-(2-(2-(2-methoxyethoxy)ethoxy)ethyl) ethanethioate (3c)


S60

4.5. GC-FID chromatograms of the prebiotic thioester formation



sample retention time 3b (min) peak area (pA*s)

1 4.428 107.07

2 4.427 101.81

3 4.428 104.05


1 4.429 124.39

2 4.429 121.55

3 4.429 121.66

S61




1 4.430 131.20

2 4.430 130.21

3 4.430 136.76


1 4.426 85.26

2 4.427 88.39

3 4.426 86.27

S62




1 4.430 126.43

2 4.430 126.80

3 4.430 122.10


1 4.415 18.18

2 4.418 19.78

3 4.418 19.58

S63




1 4.423 60.35

2 4.423 61.44

3 4.422 51.50


1 4.422 55.50

2 4.422 55.08

3 4.421 41.14

S64



1 4.418 67.62

2 4.423 76.15

3 4.423 70.98

S65

Scheme S-4, Method A

Scheme S-4, Method B


1 4.417 13.46

2 4.418 12.99

3 4.418 14.66


1 4.413 13.92

2 4.416 14.37

3 4.417 13.51

S66

Scheme S-4, Method C

Scheme S-3, Method A


1 4.450 0

2 4.450 0

3 4.418 1.21

sample retention time 3c (min) peak area (pA*s)

1 5.089 153.22

2 5.090 152.67

3 5.088 147.95

S67

Scheme S-3, Method B

Scheme S-3, Method C


1 5.092 260.85

2 5.092 254.51

3 5.091 248.65


1 5.089 188.67

2 5.090 173.98

3 5.090 194.93

S68

4.6. GC-FID chromatograms of the network combined with thioester formation

Table S-3, Entry 1

Table S-3, Entry 2


1 4.419 0.11

2 4.419 0.11

3 4.428 0.10


1 4.418 1.28

2 4.418 1.48

3 4.418 1.46

S69

Table S-3, Entry 3


1 4.418 1.21

2 4.418 0.75

3 4.418 1.01

S70

4.7. LC-MS chromatograms of authentic samples

N-acetyl cysteamine (1b) m/z = 119.95–120.15

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 0.57MA: 1942086443

0.847.66 8.388.20 8.610.95 7.577.096.826.642.67 8.631.27 6.262.891.93 9.112.17 5.733.24 9.973.54 4.523.84 9.674.12 4.76 5.445.250.42

NL:3.71E8

m/z= 119.95-120.15 MS ECB500-c5b

ECB500-c5b #61 RT: 0.57 AV: 1 SB: 240 0.00-0.51 , 0.98-2.73 NL: 3.41E8T: FTMS + p ESI Full ms [70.0000-700.0000]

100 150 200 250 300 350 400 450 500 550 600 650 700

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

120.0478

214.0890

83.0610

239.0876103.0215165.1053

138.0911 198.1848

282.1296 333.1292427.1715 450.1689355.0408 675.1624521.7238 598.6459 647.1061

S71

N,N'-(disulfanediylbis(ethane-2,1-diyl))diacetamide (1d) m/z = 236.97–237.17

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 2.65MA: 1087460145

2.943.07 3.20 8.91 9.082.44 3.490.19 5.660.69 7.27 9.535.470.82 7.87 8.183.971.17 9.781.76 5.04 7.144.452.17 4.77 6.42 7.52 8.776.685.81

NL:1.46E8

m/z= 236.97-237.17 MS JG219-11

JG199-18 #284 RT: 2.69 AV: 1 SB: 406 3.00-4.91 , 0.69-2.60 NL: 1.04E7T: FTMS + p ESI Full ms [70.0000-700.0000]

240 260 280 300 320 340 360 380 400 420 440 460 480 500

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

237.0732

282.1311

259.1485

427.1734

351.1747313.1586 368.1049 453.6292335.1796268.2466253.1805 444.2001303.1382287.2226

405.0372 419.3152381.9603 481.4226465.1290

S72

N,S-diacetyl cysteamine (3b) m/z = 161.95–162.15

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 1.69MA: 221307459

1.83

1.88

1.93 8.737.47 8.578.458.097.412.02 6.782.13 6.526.41 9.718.776.200.23 9.602.430.61 0.91 1.29 2.72 3.823.48 5.923.96 4.60 4.85 5.17

NL:3.04E7

m/z= 161.95-162.15 MS JG220-4


70 80 90 100 110 120 130 140 150 160 170 180 190 200

m/z

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

Re

lative

Ab

un

da

nce

83.0602

162.0563

138.0898100.0751 183.1722

114.0904158.0252 198.1830

120.0466 140.998890.0546

77.0387

84.0636

181.156598.5115 164.0521145.1319 196.1674171.1472 184.5378131.0802 151.0947103.0495

81.5205

134.9853110.0592 127.0741

S73

4-[[2-(Acetylamino)ethyl]thio]-4-oxobutanoic acid (5b) m/z = 219.95–220.15

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 2.29MA: 1566632049

2.582.71 2.98 6.32 6.713.42 6.971.05 2.140.42 6.150.65 4.99 7.151.52 7.50 7.924.02 9.675.14 8.804.22 8.26 9.414.73 5.91 9.04

NL:2.26E8

m/z= 219.95-220.15 MS ECB445-C4

ECB445-C4 #242 RT: 2.29 AV: 1 SB: 541 2.49-5.35 , 0.00-2.25 NL: 2.32E8T: FTMS + p ESI Full ms [70.0000-700.0000]

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

214.0863

220.0606

265.1177

83.0600120.0463

138.0893183.1716

158.0247 439.1139202.0503237.0869

258.0160

290.0229 309.1069

278.1127

342.5861 357.1422 382.6047 409.0466

S74

3-((2-acetamidoethyl)thio)-3-oxopropanoic acid (8b) m/z = 205.54–206.54

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 0.86MA: 768772457

1.18 1.32 9.580.570.42 1.84 6.232.02 9.47 9.816.452.94 3.442.42 3.94 4.43 4.78 5.00 7.106.11 6.70 9.187.727.475.22 8.608.20

NL:1.31E8

m/z= 205.54-206.54 MS CL030-Calib-20mM-1

CL030-Calib-20mM-1 #89 RT: 0.84 AV: 1 SB: 1037 0.99-10.01 , 0.00-0.80 NL: 1.01E8T: FTMS + p ESI Full ms [70.0000-700.0000]

70 80 90 100 110 120 130 140 150 160 170 180 190 200

m/z

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

Rela

tive A

bundance

206.0488

183.1749158.0275141.0008114.092090.0559 198.1858100.076577.0398 120.0484 181.1591164.0380 188.0382129.0664 145.1340 170.0276151.0971106.050786.0611

203.0851

O

O

SNH

HO

O

calculated excact mass

[M+H]+ = 206.0482

S75

4-((2-acetamidoethyl)thio)-2-hydroxy-4-oxobutanoic acid (9b) m/z = 236.04–236.06

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 0.90MA: 53114744

1.05

1.09

1.131.25 9.963.00 3.58 9.671.74 2.19 2.74 3.35

NL:7.55E6

m/z= 236.04-236.06 MS JG233-10


100 150 200 250 300 350 400 450 500 550 600 650 700

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

236.0583

281.1160

120.0479471.1094

190.0529

215.0935

258.0402 525.0292493.0913141.9584103.0216 289.9774 324.1580 425.1035449.1402

366.1321

166.1222

684.4061552.9889 607.6182 640.6078

S76

4.8. LC-MS chromatograms of thioester formation under prebiotic conditions



RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 2.05MA: 91068406

2.17

2.280.26 0.280.59 2.330.69 2.50 9.44 9.601.09 9.351.12 3.381.42 8.796.212.74 5.07 8.773.56 5.09 6.19 6.383.73 4.43 4.64 6.575.95 6.92 8.857.27 8.227.967.58 8.55

NL:1.59E7

m/z= 219.56-220.56 MS JG214-1

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 1.98MA: 316703247

2.15

2.21

2.350.69 1.73 6.770.51 2.561.09 10.006.981.57 3.67 6.33 9.342.97 3.69 7.254.65 5.104.42 6.16 9.305.27 8.785.78 7.49 8.397.94

NL:4.82E7

m/z= 219.95-220.15 MS ECB494-2-A1

sample retention time 5b (min) peak area

1 2.05 9.82E+07

2 2.00 1.48E+08

3 2.04 1.31E+08


1 1.98 3.17E+08

2 2.03 3.50E+08

3 2.03 3.04E+08

S77


RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 2.05MA: 172844401

2.22

2.24

2.350.51 9.711.85 2.46 6.671.09 1.310.36 8.813.110.52 9.606.346.20 6.724.063.773.43 5.074.21 8.775.214.69 5.96 6.98 9.297.69 7.87 8.12

NL:2.67E7

m/z= 219.95-220.15 MS ECB494-2-B1


1 2.05 1.73E+08

2 2.03 1.84E+08

3 2.03 1.60E+08

S78


RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 0.86MA: 62919194

1.011.030.40

0.52 6.201.300.18 9.821.961.75 9.417.91 8.282.37 6.18 6.472.72 2.74 8.346.653.44 6.96 9.325.145.07 5.903.76 8.43 8.823.96 4.25 7.065.22 7.48 9.22

NL:1.24E7

m/z= 205.54-206.54 MS JG214-4

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

RT: 1.54MA: 57053073

1.64

1.72

0.34 1.76 1.930.371.440.67 2.21 2.23 8.73 9.702.64

9.478.70 9.353.562.74 6.20 6.822.95 8.343.65 8.066.994.47 6.684.32 7.915.05 8.436.135.174.79 7.245.97 7.40 8.80

NL:8.06E6

m/z= 161.95-162.15 MS JG214-4


1 0.86 8.24E+07

2 0.86 8.72E+07

3 0.85 7.65E+07


1 1.54 5.71E+07

2 1.54 4.69E+07

3 1.54 4.64E+07

S79


RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.99MA: 281559510

0.40

1.210.54 6.18 6.37 8.287.806.45 7.156.131.40 8.617.721.46 5.91 9.53 9.662.672.310.20 8.63 9.072.81 4.403.18 3.64 5.544.824.17

NL:4.59E7

m/z= 205.54-206.54 MS JG218-5

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 1.70MA: 47783364

1.67

1.81

8.738.281.84

6.336.18 8.598.237.837.801.910.30 6.47 6.870.43 7.15 9.780.55 9.501.14 2.71 9.442.011.30 6.062.35 2.73 6.01

9.363.673.25 8.795.103.81 4.18 5.255.084.54 9.17

NL:6.79E6

m/z= 161.95-162.15 MS JG218-5


1 0.97 3.09E+08

2 0.99 2.82E+08

3 0.97 2.71E+08


1 1.70 4.77E+07

2 1.69 4.95E+07

3 1.65 4.00E+07

S80



RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 1.01MA: 104590916

0.31

0.33

0.39

0.43

0.45

0.47

0.490.51

1.12

0.72 1.18

1.221.32 7.401.57 7.266.43 7.476.291.80 6.502.27 7.96 8.60 8.62 9.402.32 8.365.995.84 9.582.83 3.04 3.77 5.193.94 4.533.17 4.85

NL:1.63E7

m/z= 205.54-206.54 MS JG218-3

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.91MA: 13415877

0.94

0.56 1.04

1.060.55

7.657.09 8.478.08

NL:2.15E6

m/z= 236.04-236.06 MS JG242-1


1 0.99 7.35E+07

2 0.96 1.10E+08

3 1.01 1.05E+08


1 0.91 13415877

2 0.90 11846341

3 0.92 12366159

S81



RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.88MA: 34090759

0.86

1.05

1.09

1.15

0.411.17

0.511.21

8.438.228.167.46 8.69

NL:2.85E6

m/z= 236.04-236.06 MS JG242-6

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.97MA: 7095792

0.99

1.02

0.941.07

1.14

1.18

1.22

0.59 1.26

0.56 1.27

0.53

0.51

8.698.617.65 8.42

NL:6.15E5

m/z= 236.04-236.06 MS JG242-7


1 0.88 34376793

2 0.89 39158931

3 0.86 34090759


1 0.97 7095792

2 0.98 7318094

3 0.96 7863899

S82

4.9. LC-MS chromatograms of the network combined with thioester formation

Table S-3, Entry 1

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 2.33MA: 48060218

2.29

2.27

2.480.67

0.710.62

0.75

2.54

2.600.82 2.72 5.240.48 2.822.19 5.22 8.875.323.14 9.671.601.51 3.74 8.924.913.87 4.38 8.826.485.56 5.76 7.29 7.836.506.41 8.24 8.45

NL:5.06E6

m/z= 219.95-220.15 MS ECB-508-1

RT: 0.00 - 10.01

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.94MA: 3413768

0.96

0.99

0.62

1.050.59

0.03

0.57 1.09

0.55 1.12

0.521.18

8.20 8.35

NL:5.24E5

m/z= 236.04-236.06 MS ECB-508-1


1 2.33 4.81E+07

2 2.35 3.21E+07

3 2.33 3.54E+07


1 0.94 3.41E+06

2 0.95 3.47E+06

3 0.94 3.34E+06

S83

Table S-3, Entry 2

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.96MA: 428650693

RT: 2.31MA: 24021058

0.90

0.88

0.78

0.74

0.70

0.62

0.58

0.54

2.40

2.441.31

1.45 2.500.42 2.24 3.463.44 3.48 8.882.99 4.16 4.38 9.558.860.16 5.00 9.879.294.55 5.12 5.40 6.545.83 7.726.83 8.437.16 7.88

NL:1.48E7

m/z= 219.95-220.15 MS ECB-509-A1

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 1.07MA: 206003250

0.99

0.44

0.61

1.321.41

1.53 1.95 2.160.03 2.59 2.84 6.13 8.00 8.18

NL:1.66E7

m/z= 236.04-236.06 MS ECB-509-A1


1 2.31 2.40E+07

2 2.32 2.19E+07

3 2.31 2.13E+07


1 1.07 2.06E+08

2 1.05 1.78E+08

3 1.07 2.03E+08

S84

Table S-3, Entry 3

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 0.97MA: 243856930

RT: 2.32MA: 18621876

0.93

0.82

0.75

0.72

0.70

0.64

1.13

0.59

0.57

1.17

0.54

2.291.21 2.34

2.26 2.380.52

1.240.412.43

1.390.39 3.541.43 2.21 2.49 3.663.49 4.96 5.13 8.912.85 9.700.09 9.438.874.714.254.00 5.28 5.75 8.936.84 7.28 7.816.49 8.648.356.04

NL:8.40E6

m/z= 219.95-220.15 MS ECB-509-B1

RT: 0.00 - 10.02

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

RT: 1.04MA: 184818699

1.07

0.93

0.91

0.43

0.61

1.25

1.301.42

0.04 1.70 2.13 3.872.45 8.728.39

NL:1.74E7

m/z= 236.04-236.06 MS ECB-509-B1


1 2.32 1.86E+07

2 2.31 2.58E+07

3 2.32 2.32E+07


1 1.04 1.74E+08

2 1.06 2.09E+08

3 1.08 1.87E+08

S85

4.10. GC-MS chromatograms of authentic samples

N-acetylcysteamine (1b)

N-acetylcysteamine (1b) after derivatization

-1x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

+ TIC Scan 00601006.D

Counts (%) vs. Acquisition Time (min)2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ Scan (3.717 min) 00601006.D

60.00

72.00119.10

463.40

Counts vs. Mass-to-Charge (m/z)60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

+ TIC Scan 00301001.D

Counts (%) vs. Acquisition Time (min)2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8

6x10

0

1

2

3

4

5

6

7

8

9

+ Scan (5.803 min) 00301001.D

132.00

60.00

118.0086.00

104.00

192.00147.90 265.80 403.40

Counts vs. Mass-to-Charge (m/z)60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

(N-acetylcysteamine

derivatized)

S86

4.11. GC-MS chromatograms of the network after thioester formation

Table S-3, Entry 1

1x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

+ TIC Scan 00801008.D

Counts (%) vs. Acquisition Time (min)2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+ Scan (3.392 min) 00801008.D

60.90

117.00

89.00

145.0073.00 99.90 128.8052.10 109.10 266.10177.60


6x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ Scan (3.794 min) 00801008.D

60.00

72.00119.0085.90 100.80 231.00143.10 213.20


4x10

0

0.5

1

1.5

2

2.5

3

+ Scan (4.487 min) 00801008.D

74.90

103.20

131.0058.90

145.70


(pyruvate)

Glyoxylate + pyruvate + Fe2+

(70 °C, 1 h) then addition of thiol + KHSO4 (UV-A, 3 h)

pyruvate

thiol

glyoxylate

Thiol

derivatized

HKG isocitrate

(glyoxylate)

α-ketoglutarate

(thiol)

OPD

Pyruvate adducts

S87

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+ Scan (4.730 min) 00801008.D

101.00

95.1054.80128.70112.80

67.50

158.50 284.80

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410

5x10

0

1

2

3

4

5

6

7

8

9

+ Scan (5.546 min) 00801008.D

60.00

132.00

86.00

117.90

103.90

191.00145.00 313.00 453.70335.60


5x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

+ Scan (6.164 min) 00801008.D

100.00

127.0070.90

81.80116.10 214.80172.20145.70


4x10

0

1

2

3

4

5

+ Scan (6.365 min) 00801008.D

71.00

117.10

98.80

55.0088.90 145.00

126.70

216.90173.10154.80 265.20

475.40200.80 245.20

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

+ Scan (6.820 min) 00801008.D

129.10

157.20

85.00 184.90100.6061.00

71.00138.70


(thiol derivatized +

malate)

(OPD)

(isocitrate) [M – H – 90]+

[M – H – 118]+

(HKG)

S88

Table S-3, Entry 2

1x10

0

1

2

3

4

5

6

7

8

9

+ TIC Scan 01301014.D


4x10

0

0.5

1

1.5

2

2.5

+ Scan (3.187 min) 01301014.D

70.90

98.80

55.20

85.10 128.90

143.80


4x10

0

0.5

1

1.5

2

2.5

3

+ Scan (3.218 min) 01301014.D

114.90

84.90 133.1056.80

72.1098.70


4x10

0

0.5

1

1.5

2

2.5

3

3.5

+ Scan (3.395 min) 01301014.D

117.1061.00

88.90

51.80 145.3075.10

101.20131.00


6x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

+ Scan (3.834 min) 01301014.D

60.00

71.90119.00

86.10 140.10 426.70346.40282.80209.30


pyruvate

thiol

glyoxylate

Glyoxylate + pyruvate + thiol + Fe2+

(70 °C, 1 h) then addition of K2S2O8 (70 °C, 3 h)

(pyruvate)

Thiol

derivatized

+ malate

(thiol)

malonate

levulinate

mesaconate

α-ketoglutarate

Pyruvate

adducts

(levulinate)

S89

5x10

0

1

2

3

4

5

6

+ Scan (4.073 min) 01301014.D

112.90

141.00

85.0069.00

98.90 170.90127.90 388.80


5x10

0

0.5

1

1.5

2

2.5

+ Scan (4.489 min) 01301014.D

75.00

102.90

131.00

59.1087.90

113.30 145.80304.30


5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ Scan (4.728 min) 01301014.D

100.90

143.0067.90 85.90

113.00

128.8058.10156.90


5x10

0

1

2

3

4

5

6

7

+ Scan (5.543 min) 01301014.D

59.90

132.00

86.00

118.00

104.00

190.90144.80 170.10 341.40283.40216.50


(glyoxylate)

(“mesaconates”)


malate)

S90

Table S-3, Entry 3

1x10

1

2

3

4

5

6

7

8

+ TIC Scan 01501016.D


5x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

+ Scan (3.186 min) 01501016.D

99.00

55.0070.90

128.90

144.0085.40 116.00202.40 230.30


5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+ Scan (3.220 min) 01501016.D

85.0058.00

115.10133.10

73.70 98.80


4x10

0

1

2

3

4

5

6

+ Scan (3.395 min) 01501016.D

116.9061.00

89.00

71.90 145.10436.6098.80


6x10

0

0.5

1

1.5

2

2.5

3

+ Scan (3.850 min) 01501016.D

60.00

72.00118.90

138.70 499.20196.40 304.10235.40


pyruvate

thiol

(pyruvate)

Glyoxylate + pyruvate + thiol + Fe2+

(70 °C, 3 h) then addition of K2S2O8 (70 °C, 3 h)

Thiol

derivatized

+ malate

(thiol)

(levulinate)

levulinate

malonate

mesaconate

α-ketoglutarate

Pyruvate

adducts

S91

5x10

0

1

2

3

4

5

+ Scan (4.074 min) 01501016.D

112.90

140.9084.90

69.0098.80 171.00126.40


5x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

+ Scan (4.731 min) 01501016.D

100.90

142.9068.00

85.90

58.90

157.10113.00 249.70


5x10

0

1

2

3

4

5

6

7

8

+ Scan (5.552 min) 01501016.D

60.00

131.9086.00

72.00118.00

103.90

191.20147.90 168.70 233.10


(“mesaconates”)

129.00


malate)

S92

5. Supporting references

1. Muchowska, K. B. et al. Metals promote sequences of the reverse Krebs cycle. Nat. Ecol. Evol.

1, 1716–1721 (2017).

2. Varma, S. J., Muchowska, K. B., Chatelain, P. & Moran, J. Native iron reduces CO2 to

intermediates and end-products of the acetyl CoA pathway. Nat. Ecol. Evol. 2, 1019–1024

(2018).

3. Muchowska, K. B., Varma, S. J. & Moran, J. Synthesis and breakdown of universal metabolic

precursors promoted by iron. Nature 569, 104–107 (2019).

4. Shewring, J. R. et al. Multimodal Probes: Superresolution and Transmission Electron

Microscopy Imaging of Mitochondria, and Oxygen Mapping of Cells, Using Small-Molecule

Ir(III) Luminescent Complexes. Inorg. Chem. 56, 15259–15270 (2017).

5. Fujimoto, N., Nakahodo, T. & Fujihara, H. Synthesis of Anionic Sulfonate-functionalized

Conducting Polymer Nanotubes and Selective Confinement of Cationic Gold Nanoparticles in

Their Inner Cavities via Electrostatic Interaction. Chem. Lett. 42, 1394–1396 (2013).

6. Field, L., Owen, T. C., Crenshaw, R. R. & Bryan, A. W. Organic Disulfides and Related

Substances. IV. Thiolsulfonates and Disulfides Containing 2-Aminoethyl Moieties. J. Am.

Chem. Soc. 83, 4414–4417 (1961).

7. Guntaka, N. S., Healy, A. R., Crawford, J. M., Herzon, S. B. & Bruner, S. D. Structure and

Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the

Colibactin Biosynthetic Pathway. ACS Chem. Biol. 12, 2598–2608 (2017).

8. Keddie, D. J., Grande, J. B., Gonzaga, F., Brook, M. A. & Dargaville, T. R. Amphiphilic

silicone architectures via anaerobic Thiol-Ene chemistry. Org. Lett. 13, 6006–6009 (2011).

9. Hagen, A., Poust, S., Katz, L. & Keasling, J. D. Producing adipic acid and related compounds

using hybrid polyketide synthases. 1–53 (2017).

10. Lowell, A. N. et al. Chemoenzymatic Total Synthesis and Structural Diversification of

Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring,

and C-H Functionalization. J. Am. Chem. Soc. 139, 7913–7920 (2017).

11. Palyam, N. & Majewski, M. Organocatalytic syn-Aldol Reactions of Dioxanones with (S)-

Isoserinal Hydrate: Synthesis of L-Deoxymannojirimycin and L-Deoxyidonojirimycin. J. Org.

Chem. 74, 4390–4392 (2009).

S93

12. Blaquiere, N., Shore, D. G., Rousseaux, S. & Fagnou, K. Decarboxylative ketone aldol

reactions: Development and mechanistic evaluation under metal-free conditions. J. Org. Chem.

74, 6190–6198 (2009).

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