SUEMENTARY INRMATIN - Nature · .10/.2624 SUEMENTARY INRMATIN 4 Diamidophosphate (8): Adapted from...

NATURE CHEMISTRY | www.nature.com/naturechemistry 1

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2624

Supplementary information for

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

Adam J. Coggins and Matthew W. Powner

Department of Chemistry

University College London 20 Gordon Street

London, WC1H 0AJ, UK

e-mail: [email protected]

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.



2

General experimental .................................................................................................................... 3

Diamidophosphate (8): .................................................................................................................. 4

Assessment of stability of glyceric acid 2-‐phosphate (4-‐2P): .......................................................... 4

Phosphoenol pyruvaldehyde cyanohydrin (10): ............................................................................. 4

Glycolaldehyde phosphate cyanohydrin (14): ................................................................................ 4

Glyceramide 3-‐phosphate (15): ..................................................................................................... 4

Glycolaldehyde phosphate aminonitrile (16): ................................................................................ 5

Phosphoserinamide (17): ............................................................................................................... 5

Glycolaldehyde aminonitrile: ........................................................................................................ 5

Serinamide: ................................................................................................................................... 5

Serine (5): ..................................................................................................................................... 6

Chloroacetic acid (18) from oxidation of phosphoenol pyruvate (1): .............................................. 6

Spectra .......................................................................................................................................... 7

Diamidophosphate (8) sodium salt ................................................................................................ 8

Glycolaldehyde phosphate (2-‐P) .................................................................................................... 9

Glyceraldehyde 2-‐phosphate (3-‐2P) ............................................................................................. 16

Phosphoenol pyruvaldehyde (9) .................................................................................................. 22

Phosphoenol pyruvaldehyde cyanohydrin (10) ............................................................................ 31

Phosphoenol pyruvate (1) ........................................................................................................... 33

Glycolic acid (12) ......................................................................................................................... 40

Glycolic acid phosphate (12-‐P) ..................................................................................................... 41

Glyceric acid (4) ........................................................................................................................... 41

Glyceric acid 2-‐phosphate (4-‐2P) ................................................................................................. 43

Glycolaldehyde phosphate cyanohydrin (14) ............................................................................... 45

Glyceramide 3-‐phosphate (15) .................................................................................................... 49

Glyceric acid 3-‐phosphate (4-‐3P) ................................................................................................. 52

Glycolaldehyde phosphate aminonitrile (16) ............................................................................... 57

Phosphoserine amide (17) ........................................................................................................... 60

Phosphoserine (5-‐3P) .................................................................................................................. 63

Glycolaldehyde aminonitrile ........................................................................................................ 68

Serinamide .................................................................................................................................. 69

Serine (5) ..................................................................................................................................... 71

Pyruvate (6) ................................................................................................................................ 73

Chloroacetic acid (18) .................................................................................................................. 74




3

Acetate (20) ................................................................................................................................ 75

Supplementary Table 1: Expansion of Table 1: Aqueous aldehyde oxidations. ............................. 76

General experimental

Reagents and authentic standards were obtained from commercial sources and used without further purification, unless specified. Phosphoenol pyruvate (1) and phenylphosphorodiamidate were purchased from VWR International. Glycolaldehyde (2) dimer, rac-glyceraldehyde (3), D-glyceraldehyde 3-phosphate (3-3P), D-glyceric acid (4), D-glyceric acid 2-phosphate (4-2P), D-glyceric acid 3-phosphate (4-3P), pyruvate (6), phosphoserine (5-3P), potassium ferricyanide, potassium cyanide, sodium cyanide, iron (II) chloride, hydrogen peroxide solution (30% w/w), manganese dioxide, sodium chlorite, sodium hypochlorite solution (10-15%), formaldehyde solution (37% wt. in H2O, containing 10-15% methanol stabilizer), 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS), sulfamic acid and deuterium oxide (D2O) were purchased from Sigma Aldrich. Ammonium chloride was purchased from Fluorochem. Methionine was purchased from BDH. Glycolic acid was purchased from Acros Organics. Deuterated dimethyl sulfoxide (DMSO-d6) and serine (5) were purchased from Alfa Aesar. Deionized water was obtained from an Elga Option 3 purification system. Solution pH values were measured using a Mettler Toledo Seven Compact pH meter with a Mettler Toledo InLab semi-micro pH probe or a Corning pH meter 430 with a Fischerbrand FB68801 semi-micro pH probe. The readings for D2O solutions are reported as pD, and corrected according to Covington et al.1 The readings for H2O and H2O/D2O (9:1) solutions are reported uncorrected. NMR spectra were recorded with Bruker AVANCE III 600, AVANCE 500, AVANCE III 400 and AVANCE 300 spectrometers, equipped with a Bruker 5 mm cryoprobe (600) or a gradient probe (500, 400, 300). NMR data recorded at 298 K and reported as: chemical shifts (δ) in parts per million (ppm), spin multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; aps, apparent singlet; apd, apparent doublet; apt, apparent triplet; obs., obscured), coupling constants (J) in Hertz (Hz), number of protons. Diastereotopic geminal (AB) spin systems coupled to one or two additional nuclei are reported as ABX and ABXY, respectively. Chemical shifts (δ) are reported relative to residual solvent peak, and 1Hδ and 13Cδ relative to TMS calibrated using residual solvent peak. 1H NMR spectra (H2O/D2O) are solvent suppressed (noesygppr1d) with presaturation and spoil gradients unless otherwise stated. Melting points were determined with an Electrothermal IA9000 apparatus, quoted uncorrected to the nearest ºC. Infrared spectra (IR) were recorded with a Shimadzu 100 FTIR spectrometer. Absorption maxima are reported in wavenumbers (cm-1). Mass spectra and accurate mass measurements were recorded with a Waters LCT Premier XE or Thermo Finnigan MAT 900XP instrument at the Department of Chemistry, University College London. HPLC was carried out on an Agilent 1260 system with a Polaris 5 C18-A semi-preparative (150 x 10 mm) column.




4

Diamidophosphate (8):

Adapted from method of Klement and Biberacher.1 Phenylphosphorodiamidate (10 g, 0.06 mmol) was dissolved in sodium hydroxide (4M, 30 mL) and boiled for 10 min. The solution was cooled then added drop-wise to rapidly stirring ethanol (85 mL, 0 ºC). The precipitate was collected by filtration, then desiccated over P2O5 returning the tri-hydrate of 8 (8.5 g, 85%) as a granular, white solid. 31P NMR (161 MHz, D2O) δP 13.9. m/z (CI+): 215 (17%, [M+H]+•). HRMS ([H4N2O2P+H]+•) calcd. 96.0083, found 96.0084.

Assessment of stability of glyceric acid 2-phosphate (4-2P):

Glyceric acid 2-phosphate (4-2P, 60mM,) and sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS) were dissolved in aqueous solution (H2O/D2O 9:1, pH 2 or 10) or in phosphate buffer (1M, H2O/D2O 9:1, pH 7) then incubated at 60 °C for 18 h and 1H NMR spectra were periodically acquired. No 4-2P dehydration products were detected (See Supplementary Fig. 1).

Phosphoenol pyruvaldehyde cyanohydrin (10):

Phosphoenol pyruvaldehyde (9, 200mM) and sodium cyanide (5 eq.) were dissolved in D2O and the solution was adjusted to pH 9.5 with 4M DCl. The quantitative formation of phosphoenol pyruvaldehyde cyanohydrin (10) was observed by 1H and 31P NMR spectroscopy.1H NMR (400 MHz, D2O, pD 9.5) δH 5.10 (1H, s, CH), 4.95 (1H, t, J = 1.8 Hz, CH2), 4.83 (1H, t, J = 1.8 Hz, CH2). 31P NMR (161MHz, D2O, pH 9.5) δP 0.2 (aps).

Glycolaldehyde phosphate cyanohydrin (14):

Glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (1.2 eq.) were dissolved in H2O/D2O (9:1) and the pH was adjusted (2, 7 or 9.5) with 4M HCl or 4M NaOH. Formation of 14 (94%) was observed by NMR spectroscopy at all pH values after 5 h, alongside residual 2-P (6%). 1H NMR (400 MHz, H2O/D2O 9:1, pH 7) δH 4.75 (obs., CH), 3.95 (2H, m, CH2). 13C NMR (151 MHz, H2O/D2O 9:1, pH 7) δC 120.1 (C1), 65.7 (d, J = 4.4 Hz, C3), 62.3 (d, J = 6.6 Hz, C2). 31P NMR (161 MHz, H2O/D2O 9:1, pH 7) δP 3.8 (aps).

Glyceramide 3-phosphate (15):

Glycolaldehyde phosphate (2-P, 160mM) and sodium cyanide (5 eq.) and DSS (23mM) were dissolved in phosphate buffer (750mM, H2O/D2O 9:1, pH 7) and incubated at 75 °C. The reaction was monitored NMR spectroscopy and the yield was quantified with respect to the internal DSS standard. Formation of 15 (27%), alongside 4-3P (9%), was observed after 3 d. 1H NMR (600 MHz, 750mM phosphate, H2O/D2O 9:1, pH 7) δH 4.25 (1H, dd, J = 5.2, 3.2 Hz, CH), 4.00 (1H, ABXY, J = 11.6, 7.6, 3.2 Hz, CH2), 3.92 (1H, ABXY, J = 11.6, 7.6, 5.2 Hz, CH2). 13C NMR (151 MHz, 750mM phosphate H2O/D2O 9:1, pH 7) δC 179.4 (C1); 73.5 (d, J = 7.2 Hz, C2), 67.5 (d, J = 4.4 Hz, C3). 31P NMR (161 MHz, 750 mM phosphate H2O/D2O 9:1, pH 7) δP 4.4 (t, J = 5.9 Hz). 1 Klement, R. & Biberacher, G. Das thermische Verhalten von Natriumdiamidophosphat, Darstellung von kondensierten Imidophosphaten. Z. Anorg. Allg. Chem. 285, 74–85 (1956).




5

Glycolaldehyde phosphate aminonitrile (16):

Glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (3 eq.) and ammonium chloride (5 eq.) were dissolved in H2O/D2O (9:1) and the pH was adjusted to 9.5 with 4M NaOH. The solution was incubated at room temperature with periodic analysis by NMR spectroscopy. Cyanohydrin (14) was observed to gradually convert to aminonitrile (16) over 100 h in 90% yield. 1H NMR (600 MHz, H2O/D2O 9:1, pH 9.5) δH 4.09 (1H, t, J = 5.3 Hz, CH), 3.91 (1H, ABXY, J = 10.3, 5.9, 5.3 Hz, CH2), 3.85 (1H, ABXY, J = 10.3, 5.9, 5.3 Hz, CH2). 13C NMR (151MHz, H2O/D2O 9:1, pH 9.5) δC 122.0 (C1), 65.4 (d, J = 4.0 Hz, C3), 44.5 (d, J = 8.4 Hz, C2). 31P NMR (161MHz, H2O/D2O 9:1, pH 9.5) δP 3.8 (t, J = 5.9 Hz).

Phosphoserinamide (17):

Glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (0.94 eq.), ammonium chloride (5 eq.) and DSS (22mM) were dissolved in H2O/D2O (9:1) and the pH was adjusted to 9.5 with 4M NaOH. The solution was incubated at room temperature for 3 d then analysed by NMR spectroscopy. The yield of phosphoserinamide (17), quantified with respect to the DSS internal standard, was found to be 65% (Cf. cyanide). 1H NMR (400 MHz, H2O/D2O 9:1, pH 9.5) δH 3.86 (1H, ABXY, J = 11.6, 5.4, 5.2 Hz, CH2), 3.84 (1H, ABXY, J = 11.6, 5.4, 5.2 Hz, CH2), 3.65 (1H, t, J = 5.2 Hz, CH). 13C NMR (101 MHz, H2O/D2O 9:1, pH 9.5) δC 178.5 (C1), 66.5 (d, J = 4.4 Hz, C3), 55.4 (d, J = 7.7 Hz, C2). 31P NMR (161MHz, H2O/D2O 9:1, pH 7) δP 4.1 (t, J = 5.4 Hz). The reaction of glycolaldehyde (2) with ammonium cyanide to give serinamide and then subsequent alkali hydrolysis to serine (5) was carried out for comparison of product spectra to their phosphorylated variants (2-P à 5-3P):

Glycolaldehyde aminonitrile:

Glycolaldehyde (2, 200mM), sodium cyanide (3 eq.) and ammonium chloride (5 eq.) were dissolved in H2O/D2O (9:1) and the pH was adjusted to 9.5 with 4M NaOH. The solution was incubated at room temperature with periodic analysis by 1H NMR spectroscopy. Cyanohydrin was observed to gradually convert to aminonitrile over 100 h. 1H NMR (600 MHz, H2O/D2O 9:1, pH 9.5) δH 4.01 (1H, dd, J = 5.7, 4.8 Hz, CH), 3.84 (1H, ABX, J = 11.4, 4.8 Hz, CH2), 3.75 (1H, ABX, J = 11.4, 5.7 Hz, CH2).

Serinamide:

Glycolaldehyde (2, 200mM), sodium cyanide (0.75 eq.) and ammonium chloride (5 eq.) were dissolved in H2O/D2O (9:1) and the pH was adjusted to 9.5 with 4M NaOH. The solution was incubated at room temperature for 7 d. The formation of serinamide was observed by NMR spectroscopy. 1H NMR (400 MHz, H2O/D2O 9:1, pH 9.5) δH 3.75 (1H, ABX, J = 11.4, 5.1 Hz, CH2), 3.70 (1H, ABX, J = 11.4, 5.1 Hz, CH2), 3.52 (1H, t, J = 5.1 Hz, CH). 13C NMR (101 MHz, H2O/D2O 9:1, pH 9.5) δC 178.9 (C1), 64.6 (C3), 56.5 (C2).




6

Serine (5):

Glycolaldehyde (2, 200mM), sodium cyanide (0.75 eq.) and ammonium chloride (5 eq.) were dissolved in H2O/D2O (9:1) and the pH was adjusted to 9.5 with 4M NaOH. The solution was incubated at room temperature for 7 d. The pH was then adjusted to 12 with 4M NaOH and incubation was continued at 75 °C for 30 h (the pH was re-adjusted from 10 to 12 after 26 h). The formation of 5 was observed by NMR spectroscopy and confirmed by spiking with commercially obtained 5. 1H NMR (400 MHz, H2O/D2O 9:1, pH 12) δH 3.79 (1H, ABX, J = 11.1, 4.4 Hz, CH2), 3.73 (1H, ABX, J = 11.1, 5.7 Hz, CH2), 3.39 (1H, dd, J = 5.7, 4.4 Hz, CH). 13C NMR (151 MHz, H2O/D2O 9:1, pH 12) δC 181.5 (C1), 65.7 (C3), 58.5 (C2).

Chloroacetic acid (18) from oxidation of phosphoenol pyruvate (1):

Phosphoenol pyruvate monopotassium salt (1, 100 mg, 0.49 mmol) was dissolved in H2O (7 mL) and the solution was adjusted to pH 4 with 4M NaOH. Aqueous sodium hypochlorite solution (10%, 2 eq.) was added in one portion and an aliquot, diluted with D2O (10% v/v), was analysed immediately by 1H NMR spectroscopy revealing full conversion to a single product. The solution was acidified (pH 2) with 4M HCl, transferred to a Soxhlet apparatus and extracted into hot chloroform (50 mL). The product was concentrated in vacuo to yield chloroacetic acid (18; 21 mg, 35%) as a glassy, crystalline solid. This material was found to be identical to commercial 18. 1H NMR (600 MHz, CDCl3) δH 4.14 (2H, s, CH2). 13C NMR (151 MHz, CDCl3) δC 173.3 (C1), 40.6 (C2).




7

Spectra

Fig 1: 1H NMR spectra (400 MHz, {H2O/D2O 9:1}, 3.0–7.0 ppm, pH 7)2 of glyceric acid 2-phosphate (4-2P, 60-100mM) incubated at 60 ºC for 15 h at a) pH 2 b) pH 7 (1M phosphate buffer) c) pH 10.

2 Glyceric acid 2-phosphate (4-2P) solution were neutralized (pH 7) prior to NMR acquisition. 1H NMR spectra of glyceric acid 2-phosphate (4-2P) are observed to be pH dependent: 1H NMR (400 MHz, {H2O/D2O, 9:1} pH 2) δH 4.61 (1H, obscured by solvent signal, CH), 3.87 (2H, br m, CH2). 1H NMR (400 MHz, {H2O/D2O, 9:1} pH 7) δH 4.39 (1H, ddd, J = 8.9, 5.3, 3.3 Hz, CH), 3.83 (1H, ABX, J = 11.8, 3.3 Hz, CH2), 3.74 (1H, ABX, J = 11.8, 5.3 Hz, CH2). 1H NMR (400 MHz, {H2O/D2O, 9:1} pH 10) δH 4.39 (1H, ddd, J = 8.6, 5.5, 2.9 Hz, CH), 3.84 (1H, ABX, J = 11.6, 2.9 Hz, C 3.72 (1H, ABX, J = 11.6, 5.5 Hz, CH2).




8

Diamidophosphate (8) sodium salt

Fig. 2: 31P NMR spectrum (161 MHz, D2O, pH 4, -85–90 ppm) of 8

AJC571.010.001.1r.esp

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0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

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9

Glycolaldehyde phosphate (2-P)

Fig. 3: 1H NMR spectra (400 MHz, {750mM phosphate H2O/D2O 9:1}, 3.2–6.2 ppm) of glycolaldehyde (2, 25mM) and diamidophosphate (8, 100mM) with DSS, at room temperature, pH 7. a) 0.0 h, b) 1.5 h, c) 2.5 h, d) 4.5 h, e) 8.5 h, f) 22 h. Fig. 4: 1H NMR spectra (400 MHz, {750mM phosphate, H2O/D2O, 9:1}, 3.0–6.0 ppm) of glycolaldehyde (2, 25mM) and diamidophosphate (8, 100mM) after 1.6 h at room temperature, at: a) pH 4, b) pH 5, c) pH 6 and d) pH 7.

9

Glycolaldehyde phosphate (2-P)

Fig. 3: 1H NMR spectra (400 MHz, {750mM phosphate H2O/D2O 9:1}, 3.2–6.2 ppm) of glycolaldehyde (2, 25mM) and diamidophosphate (8, 100mM) with DSS, at room temperature, pH 7. a) 0.0 h, b) 1.5 h, c) 2.5 h, d) 4.5 h, e) 8.5 h, f) 22 h. Fig. 4: 1H NMR spectra (400 MHz, {750mM phosphate, H2O/D2O, 9:1}, 3.0–6.0 ppm) of glycolaldehyde (2, 25mM) and diamidophosphate (8, 100mM) after 1.6 h at room temperature, at: a) pH 4, b) pH 5, c) pH 6 and d) pH 7.




10

Fig. 5: 1H NMR spectra (400 MHz, {750mM phosphate H2O/D2O 9:1}, 2.5–6.0 ppm) of: a) glycolaldehyde (2, 25mM) with DSS and b) after incubation with diamidophosphate (8, 100mM) at room temperature, pH 4, 4 h.




11

Fig. 6: 1H NMR spectra (600 MHz, {D2O} a) 0.0–9.5 ppm and b) 3.5–5.5 ppm) of glycolaldehyde phosphate (2-P) calcium salt.

OH

OP O

OHHO

HO




12

Fig. 7: a) 13C and b) DEPT-135 NMR spectra (151 MHz, {D2O} 10–160 ppm) of glycolaldehyde phosphate (2-P) calcium salt.




13

Fig. 8: 1H-1H COSY NMR spectrum (600 MHz, {D2O} 3.5–5.5 ppm), of glycolaldehyde phosphate (2-P) calcium salt.

Fig. 9: 1H-13C HSQC NMR spectrum (600 MHz, {D2O}) of glycolaldehyde phosphate (2-P) calcium salt.




14

Fig. 10: 1H-13C HMBC NMR spectrum (600 MHz, {D2O}) of glycolaldehyde phosphate (2-P) calcium salt. Fig. 11: 31P NMR spectrum (161 MHz, {D2O} -100–100 ppm) of glycolaldehyde phosphate (2-P) calcium salt with 31P{1H-coupled} signal overlaid.




15

Fig. 12: 1H NMR spectra (600 MHz, {D2O}, 0.0–9.5 ppm) of glycolaldehyde phosphate (2-P, 132mM) sodium salt with DSS, with expansion (3.7–5.2 ppm) overlaid.




16

Glyceraldehyde 2-phosphate (3-2P)

Fig. 13: 1H NMR spectra (400 MHz, {750mM phosphate H2O/D2O, 9:1}, 2.5–6.0 ppm) of a) glyceraldehyde (3, 25mM) with DSS and b) after incubation with diamidophosphate (8, 100mM) at room temperature, pH 4, 4 h.




17

Fig. 14: 1H NMR spectra (600 MHz, {D2O}, a) 1.0–9.5 ppm and b) 3.5–5.5 ppm) of glyceraldehyde 2-phosphate (3-2P) sodium salt.




18

Fig. 15: a) 13C and b) DEPT-135 NMR spectra (151 MHz, {D2O} 10–160 ppm) of glycerlaldehyde 2-phosphate (3-2P) sodium salt.




19

Fig. 16: 1H-1H COSY NMR spectrum (600 MHz, {D2O} 3.5 – 5.5 ppm), of glyceraldehyde 2-phosphate (3-2P) sodium salt.

Fig. 17: 1H-13C HSQC NMR spectrum (600 MHz, {D2O}) of glyceraldehyde 2-phosphate (3-2P) sodium salt.

19

Fig. 16: 1H-1H COSY NMR spectrum (600 MHz, {D2O} 3.5 – 5.5 ppm), of glyceraldehyde 2-phosphate (3-2P) sodium salt.

Fig. 17: 1H-13C HSQC NMR spectrum (600 MHz, {D2O}) of glyceraldehyde 2-phosphate (3-2P) sodium salt.




20

Fig. 18: 1H-13C HMBC NMR spectrum (600 MHz, {D2O}) of glyceraldehyde 2-phosphate (3-2P) sodium salt. Fig. 19: 31P NMR spectrum (161 MHz, {D2O} -100–100 ppm) of glyceraldehyde 2-phosphate (3-2P) sodium salt with 31P{1H-coupled} signal overlaid.

20

Fig. 18: 1H-13C HMBC NMR spectrum (600 MHz, {D2O}) of glyceraldehyde 2-phosphate (3-2P) sodium salt. Fig. 19: 31P NMR spectrum (161 MHz, {D2O} -100–100 ppm) of glyceraldehyde 2-phosphate (3-2P) sodium salt with 31P{1H-coupled} signal overlaid.




21

Fig. 20: 1H-31P HMBC NMR spectrum (400 MHz, {D2O}) of glyceraldehyde 2-phosphate (3-2P) sodium salt.




22

Phosphoenol pyruvaldehyde (9)

Fig. 21: 1H NMR spectra (400 MHz, {500mM phosphate H2O/D2O, 9:1}, 3.0–9.5 ppm) of a) glyceraldehyde 2-phosphate (3-2P, 80mM) with pentaerythritol and b) after incubation at 60 °C, pH 7, 23 h.




23

Fig. 22: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 3.0–9.5 ppm) of a) glyceraldehyde 2-phosphate (3-2P, 64mM) with pentaerythritol and b) after incubation at 60 °C, pH 10, 3 d.




24

Fig. 23: 1H NMR spectra (400 MHz, {500mM phosphate H2O/D2O, 9:1}, 3.0–9.5 ppm) of a) glycolaldehyde phosphate (2-P, 80mM) with DSS and b) after incubation with formaldehyde (500mM) at 60 °C, pH 7, 30 h. Commercial formaldehyde solution contains 10-15% methanol (3.2 ppm) stabiliser. Fig. 24: 1H NMR spectrum (600 MHz, {D2O}, 1.0–9.5 ppm) of phosphoenol pyruvaldehyde (9) sodium salt.

24

Fig. 23: 1H NMR spectra (400 MHz, {500mM phosphate H2O/D2O, 9:1}, 3.0–9.5 ppm) of a) glycolaldehyde phosphate (2-P, 80mM) with DSS and b) after incubation with formaldehyde (500mM) at 60 °C, pH 7, 30 h. Commercial formaldehyde solution contains 10-15% methanol (3.2 ppm) stabiliser. Fig. 24: 1H NMR spectrum (600 MHz, {D2O}, 1.0–9.5 ppm) of phosphoenol pyruvaldehyde (9) sodium salt.




25

Fig. 25: 1H NMR spectrum (600 MHz, {D2O} a) 0.0–10 ppm, b) 4.5–9.5 ppm with expansion overlaid and c) 4.5–5.5 ppm) of phosphoenol pyruvaldehyde (9) triethylammonium salt.

25

Fig. 25: 1H NMR spectrum (600 MHz, {D2O} a) 0.0–10 ppm, b) 4.5–9.5 ppm with expansion overlaid and c) 4.5–5.5 ppm) of phosphoenol pyruvaldehyde (9) triethylammonium salt.




26

Fig. 26: 1H-1H COSY NMR spectrum (600 MHz, {D2O}, a) 3.5–5.5 ppm and b) 5.6–5.4 ppm) of phosphoenol pyruvaldehyde (9) triethylammonium salt.




27

Fig. 27: a) 13C and b) DEPT-135 NMR spectra (151 MHz, {D2O} a) 10–120 ppm and b) 0–165 ppm) of phosphoenol pyruvaldehyde (9) triethylammonium salt.




28

Fig. 28: 1H-13C HSQC NMR spectrum (600 MHz, {D2O}) of phosphoenol pyruvaldehyde (9) triethylammonium salt. Fig. 29: 1H-13C HMBC NMR spectrum (600 MHz, {D2O}) of phosphoenol pyruvaldehyde (9) triethylammonium salt.

Hydrate HMBC correlations

Hydrate HSQC correlations




29

Fig. 30: 1H-1H NOESY NMR spectra (600 MHz, {D2O}) of a) phosphoenol pyruvaldehyde (9) triethylammonium salt and b) phosphoenol pyruvaldehyde (9) sodium salt.

Weak correlation




30

Fig. 31: 31P NMR spectrum (161 MHz, {D2O} -100–100 ppm) of phosphoenol pyruvate (9) triethylammonium salt.

Fig. 32: 1H-31P HMBC NMR spectrum (400 MHz, {D2O}) of phosphoenolpyruvate (9) triethylammonium salt.




31

Phosphoenol pyruvaldehyde cyanohydrin (10)

Fig. 33: 1H NMR spectra (400 MHz, {D2O}, 1.0–9.5 ppm) of phosphoenol pyruvaldehyde (9, 200mM) with sodium cyanide (1M) and acetate at room temperature, pD 9.5, 2 h, with expansion (4.7 – 5.3 ppm) overlaid.

Fig. 34: 31P NMR spectrum (400 MHz, {D2O}, -100–100 ppm) of phosphoenol pyruvaldehyde (9, 200mM) with sodium cyanide (1M) and acetate at room temperature, pD 9.5, 2 h.

31

Phosphoenol pyruvaldehyde cyanohydrin (10)

Fig. 33: 1H NMR spectra (400 MHz, {D2O}, 1.0–9.5 ppm) of phosphoenol pyruvaldehyde (9, 200mM) with sodium cyanide (1M) and acetate at room temperature, pD 9.5, 2 h, with expansion (4.7 – 5.3 ppm) overlaid.

Fig. 34: 31P NMR spectrum (400 MHz, {D2O}, -100–100 ppm) of phosphoenol pyruvaldehyde (9, 200mM) with sodium cyanide (1M) and acetate at room temperature, pD 9.5, 2 h.




32

Fig. 35: 1H-31P HMBC NMR spectrum (400 MHz, {D2O}) of phosphoenol pyruvaldehyde (9, 200mM) with sodium cyanide (1M) and acetate at room temperature, pD 9.5, 2 h.




33

Phosphoenol pyruvate (1)

Fig. 36: 1H NMR spectra (400 MHz, {750mM phosphate, D2O}, 2.5–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 33mM) with DSS and b) after incubation with sodium cyanide (167mM) and potassium ferricyanide (333mM) at room temperature, pD 13, 2 h.




34

Fig. 37: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 100mM) with sodium acetate (20) and b) after incubation with sodium cyanide (500mM) and manganese dioxide (20 eq.) at room temperature, pH 10.5, 2 h.




35

Fig. 38: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) the reaction of phosphoenol pyruvaldehyde (9, 100mM) with sodium cyanide (500mM) and manganese dioxide (20 eq.) at room temperature, pD 9.5, 2 h and b) after spiking with commercial phosphoenol pyruvate (1).




36

Fig. 39: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 3.0–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 25mM), b) after incubation with iron (II) chloride (25mM) and hydrogen peroxide (100mM) in phosphate buffer (750mM) at 0 - 25 °C, pH 10, 1 h followed by lyophilisation and re-dissolving in D2O and c) spike with commercial phosphoenol pyruvate (1).




37

Fig. 40: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 3.0–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 25mM) after incubation with hydrogen peroxide (50mM) in phosphate buffer (750mM) at 0 - 25 °C, pH 10, 1 h followed by evaporation and re-dissolving in D2O and c) spike with commercial phosphoenol pyruvate (1).




38

Fig. 41: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 2.5–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 70mM) with DSS b) after incubation with DMSO-d6 (140mM) and sodium chlorite (100mM) at 0 °C, pD 4, 1 h and c) spike with commercial phosphoenol pyruvate (1).




39

Fig. 42: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 2.5–9.5 ppm) of a) phosphoenol pyruvaldehyde (9, 70mM) and methionine (140mM) with DSS and b) after incubation with sodium chlorite (98mM) at 0 °C, pD 4, 1 h.




40

Glycolic acid (12)

Fig. 43: 1H NMR spectra (400 MHz, {60mM phosphate, H2O/D2O, 9:1}, -0.5–9.0 ppm) of a) glycolaldehyde (2, 70mM) with DSS, b) after incubation with DMSO-d6 (140mM) and sodium chlorite (100mM) at 0 °C, pH 4, 1 h and c) spike with commercial glycolic acid (12).




41

Glycolic acid phosphate (12-P)

Fig. 44: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 1.0–6.0 ppm) of a) glycolaldehyde phosphate (2-P, 70mM) with DSS, b) after incubation DMSO-d6 (140mM) and sodium chlorite (100mM) at 0 °C, pD 4, 2 h and c) after spiking with commercial glycolic acid phosphate (12-P).

Glyceric acid (4)




42

Fig. 45: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, -0.5–9.5 ppm) of a) glyceraldehyde (3, 70mM) with DSS and b) after incubation with DMSO-d6 (140mM) and sodium chlorite (100mM) at 0 °C, pD 4, 1 h, with expansion overlaid.




43

Glyceric acid 2-phosphate (4-2P)

Fig. 46: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) glyceraldehyde 2-phosphate (3-2P, 100mM) with DSS, b) after incubation with sodium cyanide (500mM) and sodium manganese dioxide (20 eq.) at RT, pH 10.5, 2 h and, c) after spiking with commercial glyceric acid phosphate (4-2P).




44

Fig. 47: 1H NMR spectra (400 MHz, {60mM phosphate, D2O}, 1.0–6.0 ppm) of a) glyceraldehyde 2-phosphate (3-2P, 70mM) with DSS, b) after incubation with DMSO-d6 (140mM) and sodium chlorite (100mM) at 0 °C, pD 4, 2 h and c) after spiking with commercial glyceric acid phosphate (4-2P). Fig. 48: 1H NMR spectra (400 MHz, {500mM phosphate H2O/D2O, 9:1}, 2.5–9.5 ppm) of a) glyceraldehyde phosphate (3-2P, 63mM) with DSS after incubation at 60 °C, pH 7, 5 h and b) after further incubation with DMSO-d6 (126mM) and sodium chlorite (88mM) at 0 °C, pD 4, 2 h.




45

Glycolaldehyde phosphate cyanohydrin (14)

Fig. 49: 1H NMR spectrum (600 MHz, {H2O/D2O, 9:1}, 1–9.5 ppm) of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (240mM) at room temperature, pH 7 (no solvent suppression). Fig. 50: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}, 2.9–5.9 ppm) of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (240mM) at room temperature, a) pH 2, b) pH 7 and c) pH 10.

45

Glycolaldehyde phosphate cyanohydrin (14)

Fig. 49: 1H NMR spectrum (600 MHz, {H2O/D2O, 9:1}, 1–9.5 ppm) of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (240mM) at room temperature, pH 7 (no solvent suppression). Fig. 50: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}, 2.9–5.9 ppm) of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (240mM) at room temperature, a) pH 2, b) pH 7 and c) pH 10.




46

Fig. 51: a) 13C and b) DEPT-135 NMR spectra (151 MHz, {H2O/D2O, 9:1} 0–200 ppm) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 52: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7.

46

Fig. 51: a) 13C and b) DEPT-135 NMR spectra (151 MHz, {H2O/D2O, 9:1} 0–200 ppm) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 52: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7.




47

Fig. 53: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 54: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 2.

47

Fig. 53: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 54: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 2.




48

Fig. 55: 31P NMR spectrum (161 MHz, {H2O/D2O, 9:1} -100–100 ppm), of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 56: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7.

48

Fig. 55: 31P NMR spectrum (161 MHz, {H2O/D2O, 9:1} -100–100 ppm), of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7. Fig. 56: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of glycolaldehyde phosphate cyanohydrin (14) at room temperature, pH 7.




49

Glyceramide 3-phosphate (15)

Fig. 57: 1H NMR spectra (600 MHz, {750mM phosphate H2O/D2O 9:1}) a) 1.0–9.5 ppm and b) 3.5–5.0 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d.




50

Fig. 58: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d. Fig. 59: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}), of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d.

50

Fig. 58: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d. Fig. 59: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}), of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d.




51

Fig. 60: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d. Fig. 61: 31P NMR spectrum (161 MHz, {D2O} -10–10 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d.




52

Glyceric acid 3-phosphate (4-3P)

Fig. 62: 1H NMR spectra (600 MHz, {750mM phosphate H2O/D2O, 9:1}) a) 1.0–9.5 ppm and b) 3.3–4.8 ppm of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h.




53

Fig. 63: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–9.5 ppm) of a) the reaction of glyceraldehyde 3-phosphate (3-3P, 70mM) and sodium chlorite (98mM) at 0 °C, pH 4, 0.5 h and b) after adjustment to pH 10.




54

Fig. 64: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 3.0–6.0 ppm) of a) the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h, then readjustment to pH 7. b) First spike, and c) second spike, with material from the reaction of glyceraldehyde 3-phosphate (3-3P, 70mM) and sodium chlorite (98mM) at 0 °C, pH 4, 0.5 h (adjusted to pH 7 before spiking). d) Material from 2-P cyanohydrin hydrolysis (spectra a) spiked with commercial glyceric acid 3-phosphate (4-3P).




55

Fig. 65: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h. Fig. 66: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h.

55

Fig. 65: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h. Fig. 66: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h.




56

Fig. 67: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h. Fig. 68: 31P NMR spectrum (161 MHz, {D2O} -20–20 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h with expansion (4.1–4.8 ppm) overlaid.

56

Fig. 67: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h. Fig. 68: 31P NMR spectrum (161 MHz, {D2O} -20–20 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM) and sodium cyanide (233mM) at 75 °C, pH 7, 6 d, followed by incubation at 75 °C, pH 12, 5 h with expansion (4.1–4.8 ppm) overlaid.




57

Glycolaldehyde phosphate aminonitrile (16)

Fig. 69: Stacked plot of 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 3.0–6.0 ppm) of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5 at a) 0.5 h, b) 3 h, c) 12 h, d) 24 h and e) 100 h.

Fig. 70: 1H NMR spectrum (400 MHz, {H2O/D2O, 9:1}, 1.0–9.0 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.

57

Glycolaldehyde phosphate aminonitrile (16)

Fig. 69: Stacked plot of 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 3.0–6.0 ppm) of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5 at a) 0.5 h, b) 3 h, c) 12 h, d) 24 h and e) 100 h.

Fig. 70: 1H NMR spectrum (400 MHz, {H2O/D2O, 9:1}, 1.0–9.0 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.




58

Fig. 71: 13C NMR spectrum (100 MHz, {H2O/D2O, 9:1} 0–200 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h. Fig. 72: 1H-13C HSQC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.

58

Fig. 71: 13C NMR spectrum (100 MHz, {H2O/D2O, 9:1} 0–200 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h. Fig. 72: 1H-13C HSQC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.




59

Fig. 73: 1H-13C HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.




60

Phosphoserine amide (17)

Fig. 74: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1} 1.0–9.5 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d with expansion overlaid. Fig. 75: 13C NMR spectrum (100 MHz, {H2O/D2O, 9:1} 0–200 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d.

60

Phosphoserine amide (17)

Fig. 74: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1} 1.0–9.5 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d with expansion overlaid. Fig. 75: 13C NMR spectrum (100 MHz, {H2O/D2O, 9:1} 0–200 ppm), of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d.




61

Fig. 76: 1H-13C HSQC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d. Fig. 77: 1H-13C HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d.




62

Fig. 78: 31P NMR spectrum (161 MHz, {H2O/D2O, 9:1} -100–100 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d, with 31P{1H-coupled} spectrum overlaid. Fig. 79: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d.

62

Fig. 78: 31P NMR spectrum (161 MHz, {H2O/D2O, 9:1} -100–100 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d, with 31P{1H-coupled} spectrum overlaid. Fig. 79: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (180mM) and ammonium chloride (1M) at room temperature, pH 9.5, 3 d.




63

Phosphoserine (5-3P)

Fig. 80: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}) a) 1.0–9.5 ppm and b) 3.0–6.0 ppm of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis).




64

Fig. 81: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 2.4–4.4 ppm) of a) the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) first spike, and c) second spike, with commercial phosphoserine (5-3P). Fig. 82: 13C NMR spectrum (151 MHz, {H2O/D2O 9:1} -20–220 ppm) of a) the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) commercial phosphoserine (5-3P).

64

Fig. 81: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 2.4–4.4 ppm) of a) the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) first spike, and c) second spike, with commercial phosphoserine (5-3P). Fig. 82: 13C NMR spectrum (151 MHz, {H2O/D2O 9:1} -20–220 ppm) of a) the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) commercial phosphoserine (5-3P).




65

Fig. 83: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis).




66

Fig. 84: 31P NMR spectrum (161 MHz, {H2O/D2O, 9:1} a) -100–100 ppm, b) and c) 1.5 – 8.0 ppm) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) a) 1H-decoupled, b) 1H-coupled and c) 1H-coupled, spiked with commercial phosphoserine (5-3P).




67

Fig. 85: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis). Fig. 86: Stacked plot of 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}, 4.2 - 5.2 ppm) of a) glycolaldehyde phosphate (2-P, 200mM), b) after addition of sodium cyanide (150mM) and ammonium chloride (1M) at room temperature, pH 9.5, 20 min, c) 3 h, d) 12 h, e) 24 h, f) 100 h, g) adjusted to pH 12 and incubated at 75 °C, 4 h and h) 30 h (with readjustment from pH 10 to pH 12 after 26 h).

67

Fig. 85: 1H-31P HMBC NMR spectrum (400 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde phosphate (2-P, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis). Fig. 86: Stacked plot of 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}, 4.2 - 5.2 ppm) of a) glycolaldehyde phosphate (2-P, 200mM), b) after addition of sodium cyanide (150mM) and ammonium chloride (1M) at room temperature, pH 9.5, 20 min, c) 3 h, d) 12 h, e) 24 h, f) 100 h, g) adjusted to pH 12 and incubated at 75 °C, 4 h and h) 30 h (with readjustment from pH 10 to pH 12 after 26 h).




68

Glycolaldehyde aminonitrile

Fig. 87: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 3.0–6.0 ppm) of a) glycolaldehyde (2, 200mM) and b) after incubation with sodium cyanide (600mM) and ammonium chloride (1M) at room temperature, pH 9.5, 100 h.




69

Serinamide

Fig. 88: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1}) a) 0.0–9.5 ppm and b) 3.0–6.0 ppm of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h.




70

Fig. 89: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h. Fig. 90: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h.

70

Fig. 89: 13C NMR spectrum (151 MHz, {H2O/D2O, 9:1} -20–220 ppm) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h. Fig. 90: 1H-13C HSQC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h.




71

Fig. 91: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h.

Serine (5)

Fig. 92: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1} 1.0–9.5 ppm) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (readjusted to from pH 10 to pH 12 after 26 h) with expansion overlaid.

71

Fig. 91: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h.

Serine (5)

Fig. 92: 1H NMR spectra (600 MHz, {H2O/D2O, 9:1} 1.0–9.5 ppm) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (readjusted to from pH 10 to pH 12 after 26 h) with expansion overlaid.




72

Fig. 93: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 2.0–5.0 ppm) of a) the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) first spike, and c) second spike, with commercial serine (5). Fig. 94: 13C NMR spectrum (151 MHz, {H2O/D2O 9:1} -20–220 ppm) of a) the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h and b) commercial serine (5).

72

Fig. 93: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 2.0–5.0 ppm) of a) the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h (re-adjusted to pH 7 prior to analysis) and b) first spike, and c) second spike, with commercial serine (5). Fig. 94: 13C NMR spectrum (151 MHz, {H2O/D2O 9:1} -20–220 ppm) of a) the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h and b) commercial serine (5).




73

Fig. 95: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h.

Pyruvate (6)

Fig. 96: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) phosphoenol pyruvate (1, 63mM), with pentaerythritol at room temperature, pH 9.5 and b) after incubation at 60 °C, 50 h.

73

Fig. 95: 1H-13C HMBC NMR spectrum (600 MHz, {H2O/D2O, 9:1}) of the reaction of glycolaldehyde (2, 200mM), sodium cyanide (150mM) and ammonium chloride (1M) after incubation at room temperature, pH 9.5, 100 h, then adjustment to pH 12 and incubation at 75 °C, 30 h.

Pyruvate (6)

Fig. 96: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) phosphoenol pyruvate (1, 63mM), with pentaerythritol at room temperature, pH 9.5 and b) after incubation at 60 °C, 50 h.




74

Fig. 97: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) phosphoenol pyruvate (1, 63mM), with pentaerythritol at room temperature, pH 4 and b) after incubation at 60 °C, 50 h.

Chloroacetic acid (18)

Fig. 98: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–9.5 ppm) of a) the reaction of phosphoenol pyruvaldehyde (9, 70mM) and sodium chlorite (140mM) at 0 °C, pH 4, 2 h, with DSS, b) after spiking with material from the reaction of phosphoenol pyruvate (1, 69mM) and sodium hypochlorite (140mM) at 0 °C, pH 4, 1 h and c) after spiking with commercial chloroacetic acid (18).

74

Fig. 97: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–6.0 ppm) of a) phosphoenol pyruvate (1, 63mM), with pentaerythritol at room temperature, pH 4 and b) after incubation at 60 °C, 50 h.

Chloroacetic acid (18)

Fig. 98: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–9.5 ppm) of a) the reaction of phosphoenol pyruvaldehyde (9, 70mM) and sodium chlorite (140mM) at 0 °C, pH 4, 2 h, with DSS, b) after spiking with material from the reaction of phosphoenol pyruvate (1, 69mM) and sodium hypochlorite (140mM) at 0 °C, pH 4, 1 h and c) after spiking with commercial chloroacetic acid (18).




75

Acetate (20)

Fig. 99: 1H NMR spectra (400 MHz, {H2O/D2O, 9:1}, 1.0–9.5 ppm) of a) the reaction of sodium pyruvate (6, 69mM) and sodium hypochlorite (138mM) at room temperature, pH 4 and b) after spiking with commercial acetate (20).




76

Supplementary Table 1: Expansion of Table 1: Aqueous aldehyde oxidations.

Substrate R1 R2 pH Time (h)

Oxidant (eq.)

Additive (eq.)

Product Yield (%)

9 OPO3= =CH2 13 2 K3Fe(CN)6 (10) HCN (5) 1 (13) 50 (13)

9 OPO3= =CH2 13 18 K3Fe(CN)6 (10) HCN (5) 1 (13) 42 (25)

9 OPO3= =CH2 11 19 K3Fe(CN)6 (10) HCN (5) 1 11

9 OPO3= =CH2 9 19 K3Fe(CN)6 (10) HCN (5) - -

9 OPO3= =CH2 13 18 K3Fe(CN)6 (10) - 1 173

9 OPO3= =CH2 10.5 2 MnO2 (20) HCN (5) 1 92

9 OPO3= =CH2 9.5 2 MnO2 (20) HCN (5) 1 87

9 OPO3= =CH2 9.0 2 MnO2 (20) HCN (5) 1 93

9 OPO3= =CH2 8.5 2 MnO2 (20) HCN (5) 1 89

9 OPO3= =CH2 8 2 MnO2 (20) HCN (5) 1 73

9 OPO3= =CH2 10.5 24 MnO2 (20) - 1 36

9 OPO3= =CH2 10 1 H2O2 (2) - 1 (9) 22 (12)

9 OPO3= =CH2 10 1 H2O2 (4) - 1 (9) 12 (18)

9 OPO3= =CH2 10 1 H2O2 (4) FeCl2 (1) 1 (9, 12) 30 (40, 16)

9 OPO3= =CH2 4 2 NaClO2 (1.4) H2O2 (10) 1 (18) 20 (52)

9 OPO3= =CH2 4 3 NaClO2 (1.4) H2O2 (44) 1 (18) 53 (36)

9 OPO3= =CH2 4 2 NaClO2 (1.4) H3NSO3 (10) 1 (9, 18) 27 (7, 11)

9 OPO3= =CH2 4 2 NaClO2 (1.4) NH4Cl (10) 1 (18) 40 (31)

9 OPO3= =CH2 4 2 NaClO2 (1.4) NH4Cl (20) 1 (9, 18) 35 (10, 25)

9 OPO3= =CH2 4 2 NaClO2 (1.4) DMSO (2) 1 Quant.

9 OPO3= =CH2 4 2 NaClO2 (1.4) DMS (2) 1 (18) 60 (27)

9 OPO3= =CH2 4 1 NaClO2 (1.4) DMS (10) 1 (9) 73 (25)

9 OPO3= =CH2 4 1.5 NaClO2 (1.4) Met (2) 1 99

9 OPO3= =CH2 7 2 NaClO2 (1.4) Met (2) 1 (9) Trace (76)

9 OPO3= =CH2 7 2 NaClO2 (1.4) DMSO (2) 1 (9) 11(82)

9 OPO3= =CH2 7 23 NaClO2 (1.4) DMSO (2) 1 (9) 64 (9

9 OPO3= =CH2 4 2 NaClO2 (2) - 18 Quant.

2 H OH 4 1.5 NaClO2 (1.4) DMSO (2) 12 Quant. 2-P H OPO3

= 4 2 NaClO2 (1.4) DMSO (2) 12-P Quant. 3 OH CH2OH 4 2 NaClO2 (1.4) DMSO (2) 4 93

3-2P OPO3= CH2OH 4 2.5 NaClO2 (1.4) DMSO (2) 4-2P Quant.

3-2P OPO3= CH2OH 10.5 2 MnO2 (20) HCN (5) 4-2P 25

3-3P OH CH2OPO3= 4 0.5 NaClO2 (1.4) DMSO (2) 4-3P 94

6 - - 4 0.5 NaOCl (2) - 20 Quant.

Met = methionine

3 Singh, V. N., Gangwar, M. C., Saxena, B. B. L. & Singh, M. P. Kinetics and mechanism of the oxidation of formaldehyde by hexacyanoferrate (III) ion. Can. J. Chem. 47, 1051–1056 (1968).


SUEMENTARY INRMATIN - Nature · .10/.2624 SUEMENTARY INRMATIN 4 Diamidophosphate (8): Adapted from...

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