obc supporting information 3-o-alkyl-glucose mh information lipophilic sugar nucleotide synthesis by...

SUPPORTING INFORMATION

Lipophilic sugar nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates

Malcolm P. Huestis†, Gaia A. Aish†, Joseph P. M. Hui‡, Evelyn C. Soo‡, and David L. Jakeman†,§,* †College of Pharmacy, 5968 College St., Dalhousie University, Halifax, Nova Scotia, Canada, B3H 3J5, ‡MS Metabolomics

Group, NRC-Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1, Phone: 902-426-0780. §Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J3, Fax: 902-494-1396.

GENERAL METHODS ..........................................................................................................................................................................2

CHEMICAL SYNTHESIS......................................................................................................................................................................4 GENERAL PROCEDURE FOR ALKYLATION (2B-H)...................................................................................................................................4

3-O-Butyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2b).....................................................................................................5 3-O-Hexyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2c) ....................................................................................................5 3-O-Octyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2d) ....................................................................................................6 3-O-Dodecyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2e)................................................................................................6 3-O-Hexadecyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2f) .............................................................................................7 1,2:5,6-Di-O-Isopropylidene-3-O-(2-methylpropyl)-α-D-glucofuranose (2g) .................................................................................7 1,2:5,6-Di-O-Isopropylidene-3-O-(2-ethylbutyl)-α-D-glucofuranose (2h) ......................................................................................8

GENERAL PROCEDURE FOR HYDROLYSIS AND ACETYLATION (3A-H AND 4A-H, RESPECTIVELY)...........................................................9 1,2,4,6-Tetra-O-acetyl-3-O-methyl-α-D-glucopyranose (4a)...........................................................................................................9 1,2,4,6-Tetra-O-acetyl-3-O-butyl-β-D-glucopyranose (4b) ...........................................................................................................10 1,2,4,6-Tetra-O-acetyl-3-O-hexyl-β-D-glucopyranose (4c) ...........................................................................................................10 1,2,4,6-Tetra-O-acetyl-3-O-octyl-β-D-glucopyranose (4d)............................................................................................................11 1,2,4,6-Tetra-O-acetyl-3-O-dodecyl-β-D-glucopyranose (4e) .......................................................................................................11 1,2,4,6-Tetra-O-acetyl-3-O-hexadecyl-β-D-glucopyranose (4f) ....................................................................................................12 1,2,4,6-Tetra-O-acetyl-3-O-(2-methylpropyl)-β-D-glucopyranose (4g) ........................................................................................12 1,2,4,6-Tetra-O-acetyl-3-O-(2-ethylbutyl)-β-D-glucopyranose (4h)..............................................................................................13

GENERAL PROCEDURE FOR SELECTIVE DEACETYLATION AND PHOSPHORYLATION (5A-H AND 6A-H, RESPECTIVELY)........................13 Diphenyl 2,4,6-tri-O-acetyl-3-O-methyl-α-D-glucopyranosyl-1-phosphate (6a)...........................................................................14 Diphenyl 2,4,6-tri-O-acetyl-3-O-butyl-α-D-glucopyranosyl-1-phosphate (6b)..............................................................................14 Diphenyl 2,4,6-tri-O-acetyl-3-O-hexyl-α-D-glucopyranosyl-1-phosphate (6c) .............................................................................15 Diphenyl 2,4,6-tri-O-acetyl-3-O-octyl-α-D-glucopyranosyl-1-phosphate (6d)..............................................................................16 Diphenyl 2,4,6-tri-O-acetyl-3-O-dodecyl-α-D-glucopyranosyl-1-phosphate (6e) .........................................................................16 Diphenyl 2,4,6-tri-O-acetyl-3-O-hexadecyl-α-D-glucopyranosyl-1-phosphate (6f) ......................................................................17 Diphenyl 2,4,6-tri-O-acetyl-3-O-(2-methylpropyl)-α-D-glucopyranosyl-1-phosphate (6g)...........................................................18 Diphenyl 2,4,6-tri-O-acetyl-3-O-(2-ethylbutyl)-α-D-glucopyranosyl-1-phosphate (6h)................................................................18

GENERAL PROCEDURE FOR DEPROTECTION AND ION EXCHANGE (7A-H AND 8A-H, RESPECTIVELY) ...................................................19 3-O-Methyl-α-D-glucopyranosyl phosphate diammonium salt (8a) ..............................................................................................20 3-O-Butyl-α-D-glucopyranosyl phosphate diammonium salt (8b) .................................................................................................20 3-O-Hexyl-α-D-glucopyranosyl phosphate diammonium salt (8c) ................................................................................................20 3-O-Octyl-α-D-glucopyranosyl phosphate diammonium salt (8d).................................................................................................21 3-O-Dodecyl-α-D-glucopyranosyl phosphate diammonium salt (8e) ............................................................................................21 3-O-Hexadecyl-α-D-glucopyranosyl phosphate diammonium salt (8f)..........................................................................................22 3-O-(2-Methylpropyl)-α-D-glucopyranosyl phosphate diammonium salt (8g) ..............................................................................23 3-O-(2-Ethylbutyl)-α-D-glucopyranosyl phosphate diammonium salt (8h) ...................................................................................23

1H, 13C AND 31P NMR SPECTRA OF COMPOUNDS 6A-H AND 8A-H ........................................................................................23

DETERMINATION OF PKA2 FOR Α-D-GLUCOPYRANOSYL PHOSPHATE AND COMPOUNDS 8B, 8D, 8E AND 8G ..72

ENZYMATIC SYNTHESIS .................................................................................................................................................................77 GENERAL PROCEDURE FOR NUCLEOTIDYLYLTRANSFERASE-CATALYZED SYNTHESIS OF SUGAR NUCLEOTIDES (9A-H, 10A-H) ............77

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FIGURE S1. A) SELECTED DATA FOR NUCLEOTIDYLYLTRANSFERASE-CATALYZED CONVERSION OF SUGAR-1-PHOSPHATES (8A-H) TO SUGAR NUCLEOTIDES (9A-H, 10A-H) USING A) CPS2L B) RMLA3.........................................................................................................77 TABLE S1. NUCLEOTIDYLYLTRANSFERASE-CATALYZED CONVERSION OF SUGAR-1-PHOSPHATES (8A-H) TO SUGAR NUCLEOTIDES (9A-H, 10A-H). ............................................................................................................................................................................................78

3-O-Methyl-dTDP-α-D-glucose (9a)..............................................................................................................................................79 3-O-Butyl-dTDP-α-D-glucose (9b) ................................................................................................................................................80 3-O-Hexyl-dTDP-α-D-glucose (9c)................................................................................................................................................81 3-O-Octyl-dTDP-α-D-glucose (9d) ................................................................................................................................................82 3-O-Dodecyl-dTDP-α-D-glucose (9e)............................................................................................................................................83 3-O-Hexadecyl-dTDP-α-D-glucose (9f) .........................................................................................................................................84 3-O-(2-Methylpropyl)-dTDP-α-D-glucose (9g) .............................................................................................................................85 3-O-(2-Ethylbutyl)-dTDP-α-D-glucose (9h)...................................................................................................................................86 3-O-Methyl-UDP-α-D-glucose (10a) .............................................................................................................................................87 3-O-Butyl-UDP-α-D-glucose (10b)................................................................................................................................................88 3-O-Hexyl-UDP-α-D-glucose (10c) ...............................................................................................................................................89 3-O-Octyl-UDP-α-D-glucose (10d)................................................................................................................................................90 3-O-Dodecyl-UDP-α-D-glucose (10e) ...........................................................................................................................................91 3-O-Hexadecyl-UDP-α-D-glucose (10f) ........................................................................................................................................92 3-O-(2-Methylpropyl)-UDP-α-D-glucose (10g).............................................................................................................................93 3-O-(2-Ethylbutyl)-UDP-α-D-glucose (10h)..................................................................................................................................94

General Methods

Dichloromethane and THF were dried over alumina (Innovative Technology) and stored over 3 Å molecular

sieves. All other reagents and solvents were purchased and used without further purification. All reactions

were monitored by thin layer chromatography (TLC) using Silicycle precoated silica gel plates (250 µm

thickness). The TLC plates were visualized with a potassium permanganate solution (3 g potassium

permanganate, 20 g potassium carbonate, 5 mL 5% aqueous sodium hydroxide, 300 mL distilled water), a

phenol solution (10 g phenol, 5 mL H2SO4, 95 mL ethanol), or ultraviolet light (λ = 254 nm). Unless otherwise

specified, flash chromatographic purification was performed on a Biotage SP1 HPFC. 1H, 13C and 31P NMR

spectra were obtained on a Bruker AVANCE-500 NMR Spectrometer operating at frequencies of 500.13 MHz,

125.76 MHz, and 202.45 MHz, respectively. Assignments are based on COSY and HSQC 2D NMR

experiments. For chloroform-d, 1H NMR chemical shifts are reported as δ in units of parts per million (ppm)

downfield from tetramethylsilane (δ 0.0) and 13C NMR chemical shifts are reported as δ in units of parts per

million (ppm) relative to the residual solvent signal of chloroform-d (δ 77.16). For methanol-d4, 1H and 13C

NMR chemical shifts are reported as δ in units of parts per million (ppm) relative to the residual solvent signal

of methanol-d4 (δ 3.31 and δ 49.00, respectively). 31P NMR chemical shifts are reported as δ in units of parts

per million (ppm) relative to 85% H3PO4. Enzymatic reactions were monitored by HPLC performed on a

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Hewlett Packard Series 1050 instrument with an Agilent Zorbax 5µm Rx-C18 column (150 cm x 4.6 cm).

Compounds with a nucleotide base chromophore were monitored using a UV detector (λ = 254 nm). The linear

gradient used was 90/10 A/B to 40/60 A/B over 8.0 min, followed by a plateau at 40/60 A/B from 8.0 to 10.0

min at 1.0 mL/min where A is an aqueous buffer containing 12 mM NBu4Br, 10 mM KH2PO4, and 5% HPLC

grade CH3CN (pH 4.9) and B is HPLC CH3CN except for assays containing compound 8f, whereby the gradient

was run to 80% CH3CN instead of 60%. High resolution mass spectra were recorded on a micrOTOF

instrument (Bruker Dalton) running in negative ion mode (ESI). Low resolution mass spectra were recorded on

an LCQDuo ion trap instrument (Thermo Finnigan) running in positive mode (ESI).

An Agilent 1100 LC system was coupled to an Applied Biosystems-MDS SCIEX hybrid triple

quadrupole linear ion trap (QTRAP 4000) mass spectrometer equipped with a Turbo V source for electrospray

ionization for the ESI-MS/MS experiments. The sample was analyzed by flow-injection analysis in 75:25 (v/v)

acetonitrile:de-ionized water using a flow-rate of 200 µL/min. Precursor ion scanning of m/z 323 ([UMP – H]-)

and m/z 383 ([TDP – H]-) were used initially to selectively detect for uridine- and thymidine-linked sugar

nucleotides, respectively, in the sample. ESI-MS/MS analysis in the enhanced product ion (EPI) mode was then

performed to confirm the presence of the expected synthetic sugar nucleotide. The mass spectrometer settings

for precursor ion scanning were: ionspray voltage 4.5 kV; mass range Q1 m/z 300 – 800; scan time 5 sec, Q1

and Q3 set to unit resolution. For the ESI-MS/MS experiments: ionspray voltage 4.5 kV; Q3 m/z 100–800; scan

speed 1000 amu/sec; trap fill time was set to dynamic; Q1 and Q3 set to unit resolution. All acquisitions were

made in the negative mode.

pKa values were measured in the following way: Using an IQ Scientific Instruments IQ150 fitted with

an ISFET probe, a 0.01 M solution of the diammonium salt was adjusted to pH 10 with 0.2 M NaOH. Titration

was done with 5 uL aliquots of 0.2 M HCl until pH 2, and the pKa values were determined by plotting in GraFit

5.0.4 (Erithacus Software Limited).

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Chemical Synthesis

NaH

RBr

DMF

1) Amberlite H+, 60°C

ORO

AcO

OAc

AcO O PO

OPhOPh

2) Ac2O, py

2) ClPO(OPh)2

1) H2, PtO21:1 EtOAc/EtOH

2) MeOH/H2O/NEt32:2:1

ORO

AcO

OAc

OAc

50°C

ORO

HO

OH

HO O PO

OO NH4

NH43) Amberlite H+, NH4OH

1

1) hydrazine acetate

OAc

O

OO

OHOO

O

OO

OROO

DMAP, CH2Cl2

DMF

2b R = (CH2)3CH3 (62%)2c R = (CH2)5CH3 (68%)2d R = (CH2)7CH3 (76%)2e R = (CH2)11CH3 (71%)2f R = (CH2)15CH3 (70%)2g R = CH2CH(CH3)2 (37%)2h R = CH2CH(CH2CH3)2 (17%)

4a R = CH3 (100%)4b R = (CH2)3CH3 (89%)4c R = (CH2)5CH3 (86%)4d R = (CH2)7CH3 (61%)4e R = (CH2)11CH3 (32%)4f R = (CH2)15CH3 (53%)4g R = CH2CH(CH3)2 (65%)4h R = CH2CH(CH2CH3)2 (98%)



Overall yields: 8a (20%), 8b (14%), 8c (21%), 8d (21%), 8e (3%), 8f (4%), 8g (3%), 8h (6%).

General Procedure for Alkylation (2b-h)

NaHRBr

DMF

1

O

OO

OHOO

O

OO

OROO

2b R = (CH2)3CH3 (62%)2c R = (CH2)5CH3 (68%)2d R = (CH2)7CH3 (76%)2e R = (CH2)11CH3 (71%)2f R = (CH2)15CH3 (70%)2g R = CH2CH(CH3)2 (37%)2h R = CH2CH(CH2CH3)2 (17%)

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To a stirring solution of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (1) (1.5 g, 5.76 mmol) in anhydrous

DMF (15 mL) was added sodium hydride (0.42 g, 17 mmol) at 0ºC. After stirring at 0ºC for 10 min, the alkyl

bromide (8.64 mmol) was added under a nitrogen atmosphere, and the mixture was stirred for another 30 min at

0ºC followed by 30 min at rt. The mixture was diluted with H2O (50 mL) and extracted with dichloromethane

(2 x 25 mL). The combined organic extracts were washed with brine (10 mL) and dried (Na2SO4), filtered and

concentrated. Elution through a silica plug afforded the title compounds (2b-h).

3-O-Butyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2b)1

Reaction with 1-bromobutane (0.93 mL, 8.64 mmol) followed by chromatographic purification

(hexanes/EtOAc, 95:5) furnished compound 2b as a colorless liquid (1.12 g, 62 %); RF = 0.48 (hexanes/EtOAc,

85:15); 1H NMR (CDCl3) δ 5.87 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.52 (d, 1H, J1,2 = 3.7 Hz, H-2), 4.31 (dd, 1H, J5,6 =

13.5 Hz, J4,5 = 6.2 Hz, H-5), 4.13 (dd, 1H, J4,5 = 7.4 Hz, J3,4 = 3.0 Hz, H-4), 4.08 (ddd, 1H, J5,6a = 8.5 Hz, 2J6a,6b

= 6.2 Hz, 4J4,6a = 0.7 Hz, H-6a), 3.98 (ddd, 1H, J5,6b = 8.5 Hz, 2J6a,6b = 6.0 Hz, 4J4,6b = 0.6 Hz, H-6b), 3.85 (d,

1H, J3,4 = 3.0 Hz, H-3), 3.56 (m, 2H, OCH2), 1.54 (m, 2H, OCH2CH2), 1.50, 1.42, 1.35, 1.32 (s, 12H, CH3 x 4),

1.38 (m, 2H, OCH2CH2CH2), 0.91 (t, 3H, J = 7.5 Hz, (CH2)3CH3); 13C NMR (CDCl3) δ 111.8, 109.0 (C(CH3)2

x 2) 105.4 (C-1), 82.7 (C-2), 82.3 (C-3), 81.4 (C-4), 72.7 (C-5), 70.5 (OCH2), 67.4 (C-6), 31.9 (OCH2CH2),

27.0, 26.9, 26.4, 25.5 (C(CH3)2 x 2), 19.4 (OCH2CH2CH2), 14.0 ((CH2)3CH3); LRMS m/z calcd for C16H28O6

[M+Na]+: 339.2. Found 339.2.

3-O-Hexyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2c)1

Reaction with 1-bromohexane (1.21 mL, 8.64 mmol) followed by chromatographic purification

(hexanes/EtOAc, 91:9) furnished compound 2c as a colorless liquid (1.35 g, 68 %); RF = 0.22 (hexanes/EtOAc,


13.5 Hz, J4,5 = 6.1 Hz, H-5), 4.13 (dd, 1H, J4,5 = 7.4 Hz, J3,4 = 3.1 Hz, H-4), 4.08 (ddd, 1H, J5,6a = 8.4 Hz, 2J6a,6b 1 Ikekawa, T.; Irinoda, K.; Saze, K.; Katori, T.; Matsuda, H.; Ohkawa, M.; Kosik, M. Chem. Pharm. Bull. 1987, 35, 2894-2899.

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= 6.2 Hz, 4J4,6a = 0.7 Hz, H-6a), 3.98 (dd, 1H, J5,6b = 8.7 Hz, 2J6a,6b = 6.0 Hz, H-6b), 3.85 (d, 1H, J3,4 = 3.1 Hz,

H-3), 3.55 (m, 2H, OCH2), 1.55 (m, 2H, OCH2CH2), 1.50, 1.42, 1.35, 1.32 (s, 12H, CH3 x 4), 1.38-1.24 (m, 6H,

OCH2CH2(CH2)3CH3), 0.89 (t, 3H, J = 6.7 Hz, (CH2)5CH3); 13C NMR (CDCl3) δ 111.8, 109.0 (C(CH3)2 x 2)

105.4 (C-1), 82.7 (C-2), 82.3 (C-3), 81.4 (C-4), 72.7 (C-5), 70.9 (OCH2), 67.4 (C-6), 31.7 (OCH2CH2), 29.8

(OCH2CH2CH2), 27.0, 26.9, 26.4, 25.5 (C(CH3)2 x 2), 25.9 (O(CH2)3CH2CH2CH3), 22.7 (O(CH2)4CH2CH3),

14.2 ((CH2)5CH3); LRMS m/z calcd for C18H32O6 [M+Na]+: 367.2. Found 367.2.

3-O-Octyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2d)1

Reaction with 1-bromooctane (1.49 mL, 8.64 mmol) followed by chromatographic purification

(hexanes/EtOAc, 93:7) furnished compound 2d as a colorless liquid (1.64 g, 76 %); RF = 0.38 (hexanes/EtOAc,


13.5 Hz, J4,5 = 6.1 Hz, H-5), 4.13 (dd, 1H, J4,5 = 7.5 Hz, J3,4 = 3.1 Hz, H-4), 4.08 (dd, 1H, J5,6a = 8.5 Hz, 2J6a,6b =

6.3 Hz, H-6a), 3.98 (dd, 1H, J5,6b = 8.5 Hz, 2J6a,6b = 6.0 Hz, H-6b), 3.85 (d, 1H, J3,4 = 3.2 Hz, H-3), 3.55 (m, 2H,

OCH2), 1.55 (m, 2H, OCH2CH2), 1.49, 1.42, 1.35, 1.32 (s, 12H, CH3 x 4), 1.38-1.21 (m, 10H,


105.4 (C-1), 82.7 (C-2), 82.3 (C-3), 81.4 (C-4), 72.7 (C-5), 70.9 (OCH2), 67.4 (C-6), 32.0, 29.5, 29.4, 26.2, 22.8

(OCH2CH2(CH2)5CH3), 29.9 (OCH2CH2(CH2)5CH3), 27.0, 26.9, 26.4, 25.5 (C(CH3)2 x 2), 14.2 ((CH2)7CH3);

LRMS m/z calcd for C20H36O6 [M+Na]+: 395.2. Found 395.3.

3-O-Dodecyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2e)1

Reaction with 1-bromododecane (2.07 mL, 8.64 mmol) followed by chromatographic purification

(hexanes/EtOAc, 93:7) furnished compound 2e as a colorless liquid (1.75 g, 71 %); RF = 0.19 (hexanes/EtOAc,



6.3 Hz, H-6a), 3.98 (dd, 1H, J5,6b = 8.5 Hz, 2J6a,6b = 6.0 Hz, H-6b), 3.85 (d, 1H, J3,4 = 3.1 Hz, H-3), 3.55 (m, 2H,

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OCH2), 1.55 (m, 2H, OCH2CH2), 1.49, 1.42, 1.35, 1.31 (s, 12H, CH3 x 4), 1.37-1.21 (m, 18H,


105.4 (C-1), 82.7 (C-2), 82.3 (C-3), 81.4 (C-4), 72.7 (C-5), 70.9 (OCH2), 67.4 (C-6), 32.0, 29.9, 29.8, 29.8,

29.7, 29.7, 29.5, 29.5, 26.4, 22.8 (OCH2(CH2)10CH3), 27.0, 26.9, 26.2, 25.5 (C(CH3)2 x 2), 14.2 ((CH2)11CH3);


3-O-Hexadecyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2f)1

Reaction with 1-bromohexadecane (3.52 mL, 11.5 mmol) followed by chromatographic purification

(hexanes/EtOAc, 90:10) furnished compound 2f as a colorless liquid (1.95 g, 70 %); RF = 0.40 (hexanes/EtOAc,

90:10); 1H NMR (CDCl3) δ 5.87 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.52 (d, 1H, J1,2 = 3.7 Hz, H-2), 4.30 (ddd, 1H, J4,5

= 7.3 Hz, J5,6a = 6.2 Hz, J5,6b = 6.2 Hz, H-5), 4.13 (dd, 1H, J4,5 = 7.4 Hz, J3,4 = 3.1 Hz, H-4), 4.08 (dd, 1H, J5,6a =

8.5 Hz, 2J6a,6b = 6.2 Hz, H-6a), 3.97 (dd, 1H, J5,6b = 8.5 Hz, 2J6a,6b = 6.0 Hz, H-6b), 3.85 (d, 1H, J3,4 = 3.1 Hz, H-

3), 3.55 (m, 2H, OCH2), 1.55 (m, 2H, OCH2CH2), 1.49, 1.42, 1.35, 1.31 (s, 12H, CH3 x 4), 1.37-1.21 (m, 26H,


105.4 (C-1), 82.7 (C-2), 82.3 (C-3), 81.4 (C-4), 72.7 (C-5), 70.9 (OCH2), 67.4 (C-6), 32.1, 29.9, 29.9, 29.9,

29.8, 29.8, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 26.2, 22.8 (OCH2(CH2)14CH3), 27.0, 26.9, 26.4, 25.5 (C(CH3)2 x

2), 14.2 ((CH2)15CH3); LRMS m/z calcd for C28H52O6 [M+Na]+: 507.4. Found 507.4.

1,2:5,6-Di-O-Isopropylidene-3-O-(2-methylpropyl)-α-D-glucofuranose (2g)

Reaction with 1-bromo-2-methylpropane (2.51 mL, 23.0 mmol) followed by chromatographic purification

(hexanes/EtOAc, 90:10) furnished compound 2g as a colorless liquid (0.67 g, 37 %); RF = 0.86

(hexanes/EtOAc, 66:33); 1H NMR (CDCl3) δ 5.87 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.52 (d, 1H, J1,2 = 3.8 Hz, H-2),

4.33 (dd, 1H, J5,6 = 13.5 Hz, J4,5 = 6.2 Hz, H-5), 4.14 (dd, 1H, J4,5 = 7.4 Hz, J3,4 = 3.1 Hz, H-4), 4.08 (dd, 1H,

J5,6a = 8.5 Hz, 2J6a,6b = 6.2 Hz, H-6a), 3.97 (dd, 1H, J5,6b = 8.5 Hz, 2J6a,6b = 6.2 Hz, H-6b), 3.84 (d, 1H, J3,4 = 3.2

Hz, H-3), 3.37 (dd, 1H, J = 6.5 Hz, 2J = 9.0 Hz, OCH2a), 3.28 (dd, 1H, J = 6.5 Hz, 2J = 9.0 Hz, OCH2b), 1.84

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(nonet, 1H, J = 6.7 Hz, OCH2CH(CH3)2), 1.50, 1.42, 1.35, 1.32 (s, 12H, CH3 x 4), 0.91 (d, 3H, J = 3.8 Hz,

CH3a), 0.90 (d, 3H, J = 3.8 Hz, CH3b); 13C NMR (CDCl3) δ 111.7, 108.9 (C(CH3)2 x 2) 105.3 (C-1), 82.4 (C-2),

82.3 (C-3), 81.3 (C-4), 77.3 (OCH2), 72.6 (C-5), 67.3 (C-6), 28.6 (OCH2CH), 26.9, 26.8, 26.3, 25.4 (C(CH3)2 x

2), 19.3, 19.2 (OCH2CH(CH3)2); LRMS m/z calcd for C16H28O6 [M+Na]+: 339.2. Found 339.1.

1,2:5,6-Di-O-Isopropylidene-3-O-(2-ethylbutyl)-α-D-glucofuranose (2h)

Reaction with 1-bromo-2-ethylbutane (1.61 mL, 11.5 mmol) followed by chromatographic purification

(hexanes/EtOAc, 91:9) furnished compound 2h as a colorless liquid (0.34 g, 17 %); RF = 0.67 (hexanes/EtOAc,



6.3 Hz, H-6a), 3.96 (dd, 1H, J5,6b = 8.4 Hz, 2J6a,6b = 6.2 Hz, H-6b), 3.83 (d, 1H, J3,4 = 3.1 Hz, H-3), 3.50 (dd,

1H, J = 5.3 Hz, 2J = 9.0 Hz, OCH2a), 3.40 (dd, 1H, J = 5.4 Hz, 2J = 9.0 Hz, OCH2b), 1.50, 1.42, 1.34, 1.32 (s,

12H, CH3 x 4), 1.45-1.28 (m, 5H, OCH2CH(CH2CH3)2), 0.88 (t, 3H, J = 7.4 Hz, CH3a), 0.90 (t, 3H, J = 7.4 Hz,

CH3b); 13C NMR (CDCl3) δ 111.9, 109.0 (C(CH3)2 x 2) 105.5 (C-1), 82.5 (C-2), 82.5 (C-3), 81.6 (C-4), 72.8

(OCH2), 72.7 (C-5), 67.5 (C-6), 41.5 (OCH2CH), 27.0, 26.9, 26.4, 25.5 (C(CH3)2 x 2), 23.5, 23.5

(OCH2CH(CH2CH3)2), 11.3, 11.2 (OCH2CH(CH2CH3)2); LRMS m/z calcd for C18H32O6 [M+Na]+: 367.2.

Found 367.2.

S9

General Procedure for Hydrolysis and Acetylation (3a-h and 4a-h, respectively)

ORO

HO

OH

OH OH

Amberlite H+

60°C

Ac2Opyr O

ROAcO

OAc

OAc

2b R = (CH2)3CH32c R = (CH2)5CH32d R = (CH2)7CH32e R = (CH2)11CH32f R = (CH2)15CH32g R = CH2CH(CH3)22h R = CH2CH(CH2CH3)2

3a R = CH33b R = (CH2)3CH33c R = (CH2)5CH33d R = (CH2)7CH33e R = (CH2)11CH33f R = (CH2)15CH33g R = CH2CH(CH3)23h R = CH2CH(CH2CH3)2

OAc

O

OO

OROO


To a solution of 3-O-alkyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (2b-h) (1.5 g) in 5:1 MeOH/H2O (30

mL) was added Amberlite IR-120 PLUS(H) ion exchange resin (45 g) and the reaction was stirred overnight at

60ºC. The reaction mixture was then filtered and concentrated to dryness. The hydrolyzed intermediate (3b-h)

was immediately reacted without further purification. Under a nitrogen atmosphere, a solution of the 3-O-alkyl-

D-glucopyranose (3a-h) in anhydrous pyridine (20 mL) was charged with acetic anhydride (3.0 mL) and stirred

overnight at rt. This mixture was then diluted with H2O (50 mL) and extracted with dichloromethane (2 x 25

mL). The combined organic extracts were washed with brine (10 mL) and dried (Na2SO4), filtered and

concentrated to afford the acetylated product (4a-h).

1,2,4,6-Tetra-O-acetyl-3-O-methyl-α-D-glucopyranose (4a)

Compound 4a was obtained as a colorless liquid (2.80 g, 100 %); RF = 0.46 (hexanes/EtOAc, 67:33); 1H NMR

(CDCl3) δ 6.29 (d, 1H, J1,2 = 3.7 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.2 Hz, J3,4 = 9.6 Hz, H-4), 5.00 (dd, 1H, J2,3 =

9.9 Hz, J1,2 = 3.6 Hz, H-2), 4.20 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a = 4.5 Hz, H-6a), 4.07 (dd, 1H, 2J6a,6b = 12.3 Hz,

J5,6b = 2.3 Hz, H-6b), 4.01 (ddd, 1H, J4,5 = 10.2 Hz, J5,6a = 4.2 Hz, J5,6b = 2.2 Hz, H-5), 3.72 (dd, 1H, J2,3 = 9.8

Hz, J3,4 = 9.8 Hz, H-3), 3.47 (s, 3H, CH3), 2.17, 2.11, 2.09, 2.07 (s, 12H, C(O)CH3 x 4); 13C NMR (CDCl3) δ

170.8, 169.7, 169.4, 168.9 (C(O)CH3 x 4), 89.6 (C-1), 78.3 (C-3), 71.2 (C-2), 70.4 (C-5), 69.1 (C-4), 61.9 (C-6),

S10

60.2 (CH3), 21.0, 20.9, 20.8, 20.8 (C(O)CH3 x 4); LRMS m/z calcd for C15H22O10 [M+Na]+: 385.1. Found

385.1.

1,2,4,6-Tetra-O-acetyl-3-O-butyl-β-D-glucopyranose (4b)

Compound 4b was obtained as a colorless liquid (1.74 g, 89 %); RF = 0.46 (hexanes/EtOAc, 67:33); 1H NMR




Hz, J3,4 = 9.5 Hz, H-3), 3.54 (m, 2H, OCH2), 2.11, 2.09, 2.08, 2.08 (s, 12H, C(O)CH3 x 4), 1.46 (m, 2H,

OCH2CH2), 1.30 (m, 2H, OCH2CH2CH2), 0.88 (t, 3H, J = 7.3 Hz, (CH2)3CH3); 13C NMR (CDCl3) δ 170.9,

169.4, 169.4, 169.2 (C(O)CH3 x 4), 92.2 (C-1), 80.4 (C-3), 73.2 (C-5), 72.4 (OCH2), 71.7 (C-2), 69.2 (C-4),

62.0 (C-6), 32.3 (OCH2CH2), 21.0, 20.9, 20.9, 20.9 (C(O)CH3 x 4), 19.1 (OCH2CH2CH2), 14.0 (O(CH2)3CH3);


1,2,4,6-Tetra-O-acetyl-3-O-hexyl-β-D-glucopyranose (4c)

Compound 4c was obtained as a white solid (1.62 g, 86 %); RF = 0.26 (hexanes/EtOAc, 80:20); 1H NMR




Hz, J3,4 = 9.4 Hz, H-3), 3.53 (m, 2H, OCH2), 2.11, 2.09, 2.08, 2.08 (s, 12H, C(O)CH3 x 4), 1.46 (p, 2H, J = 7.5

Hz, OCH2CH2), 1.26 (m, 6H, OCH2CH2(CH2)3CH3), 0.88 (t, 3H, J = 6.9 Hz, O(CH2)5CH3); 13C NMR (CDCl3)

δ 170.9, 169.4, 169.3, 169.2 (C(O)CH3 x 4), 92.2 (C-1), 80.4 (C-3), 73.2 (C-5), 72.8 (OCH2), 71.6 (C-2), 69.1

(C-4), 61.9 (C-6), 31.7, 25.7, 22.7 (OCH2CH2(CH2)3CH3), 30.3 (OCH2CH2(CH2)3CH3), 21.0, 20.9, 20.9, 20.9

(C(O)CH3 x 4), 14.1 (O(CH2)5CH3); LRMS m/z calcd for C20H32O10 [M+Na]+: 455.2. Found 455.1.

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1,2,4,6-Tetra-O-acetyl-3-O-octyl-β-D-glucopyranose (4d)2

Diethyl ether was used instead of dichloromethane as an extraction solvent en route to compound 4d, a white

solid (1.21 g, 61 %); RF = 0.32 (hexanes/EtOAc, 75:25); 1H NMR (CDCl3) δ 5.64 (d, 1H, J1,2 = 8.3 Hz, H-1),

5.08 (dd, 1H, J2,3 = 9.3 Hz, J1,2 = 8.2 Hz, H-2), 5.08 (dd, 1H, J4,5 = 9.6 Hz, J3,4 = 9.6 Hz, H-4), 4.22 (dd, 1H,

2J6a,6b = 12.4 Hz, J5,6a = 4.9 Hz, H-6a), 4.09 (dd, 1H, 2J6a,6b = 12.2 Hz, J5,6b = 2.2 Hz, H-6b), 3.72 (ddd, 1H, J4,5

= 9.9 Hz, J5,6a = 4.9 Hz, J5,6b = 2.2 Hz, H-5), 3.57 (dd, 1H, J2,3 = 9.3 Hz, J3,4 = 9.3 Hz, H-3), 3.53 (m, 2H,

OCH2), 2.10, 2.08, 2.08, 2.07 (s, 12H, C(O)CH3 x 4), 1.46 (m, 2H, OCH2CH2), 1.33-1.20 (m, 10H,

OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J = 7.1 Hz, (CH2)7CH3); 13C NMR (CDCl3) δ 170.8, 169.4, 169.3, 169.1

(C(O)CH3 x 4), 92.2 (C-1), 80.4 (C-3), 73.2 (C-5), 72.7 (OCH2), 71.7 (C-2), 69.2 (C-4), 62.0 (C-6), 31.9, 29.5,

29.3, 26.1, 22.7 (OCH2CH2(CH2)5CH3), 30.3 (OCH2CH2(CH2)5CH3), 21.0, 20.9, 20.9, 20.9 (C(O)CH3 x 4), 14.2

(O(CH2)7CH3); LRMS m/z calcd for C22H36O10 [M+Na]+: 483.2. Found 483.2.

1,2,4,6-Tetra-O-acetyl-3-O-dodecyl-β-D-glucopyranose (4e)2

Diethyl ether was used instead of dichloromethane as an extraction solvent and the reaction mixture was

purified by column chromatography (hexanes/EtOAc, 75:25). Compound 4e was obtained as a white solid

(0.58 g, 32 %); RF = 0.21 (hexanes/EtOAc, 80:20); 1H NMR (CDCl3) δ 5.64 (d, 1H, J1,2 = 8.3 Hz, H-1), 5.09

(dd, 1H, J2,3 = 9.3 Hz, J1,2 = 9.4 Hz, H-2), 5.07 (dd, 1H, J4,5 = 9.5 Hz, J3,4 = 9.5 Hz, H-4), 4.22 (dd, 1H, 2J6a,6b =

12.5 Hz, J5,6a = 5.1 Hz, H-6a), 4.10 (dd, 1H, 2J6a,6b = 12.4 Hz, J5,6b = 2.1 Hz, H-6b), 3.72 (ddd, 1H, J4,5 = 9.9 Hz,

J5,6a = 4.8 Hz, J5,6b = 2.3 Hz, H-5), 3.56 (dd, 1H, J2,3 = 9.4 Hz, J3,4 = 9.4 Hz, H-3), 3.53 (m, 2H, OCH2), 2.10,

2.08, 2.08, 2.07 (s, 12H, C(O)CH3 x 4), 1.46 (m, 2H, OCH2CH2), 1.33-1.19 (m, 18H, OCH2CH2(CH2)9CH3),

0.88 (t, 3H, J = 7.0 Hz, (CH2)11CH3); 13C NMR (CDCl3) δ 170.9, 169.4, 169.3, 169.1 (C(O)CH3 x 4), 92.2 (C-

1), 80.4 (C-3), 73.2 (C-5), 72.8 (OCH2), 71.7 (C-2), 69.2 (C-4), 62.0 (C-6), 32.1, 29.8, 29.8, 29.8, 29.7, 29.6,

29.5, 26.1, 22.8 (OCH2CH2(CH2)9CH3), 30.3 (OCH2CH2(CH2)9CH3), 21.0, 20.9, 20.9, 20.9 (C(O)CH3 x 4), 14.2

(O(CH2)11CH3); LRMS m/z calcd for C26H44O10 [M+Na]+: 539.3. Found 539.2.

2 Miethchen, R.; Gabriel, T.; Peters, D.; Holz, J.; Michalik, M. Carbohydr. Res. 1991, 214, 331-6.

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1,2,4,6-Tetra-O-acetyl-3-O-hexadecyl-β-D-glucopyranose (4f)2

Hydrolysis of the isopropylidene functionality was performed at 85ºC and chloroform was used instead of

dichloromethane as an extraction solvent for the acetylation. Compound 4f was obtained as a white solid (0.80

g, 53 %); RF = 0.62 (hexanes/EtOAc, 67:33); 1H NMR (CDCl3) δ 5.64 (d, 1H, J1,2 = 8.2 Hz, H-1), 5.08 (dd, 1H,

J2,3 = 9.3 Hz, J1,2 = 9.4 Hz, H-2), 5.07 (dd, 1H, J4,5 = 9.5 Hz, J3,4 = 9.5 Hz, H-4), 4.22 (dd, 1H, 2J6a,6b = 12.4 Hz,

J5,6a = 4.9 Hz, H-6a), 4.10 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6b = 2.3 Hz, H-6b), 3.72 (ddd, 1H, J4,5 = 9.9 Hz, J5,6a =

4.9 Hz, J5,6b = 2.3 Hz, H-5), 3.56 (dd, 1H, J2,3 = 9.3 Hz, J3,4 = 9.3 Hz, H-3), 3.52 (m, 2H, OCH2), 2.10, 2.08,

2.08, 2.07 (s, 12H, C(O)CH3 x 4), 1.46 (m, 2H, OCH2CH2), 1.33-1.19 (m, 26H, OCH2CH2(CH2)13CH3), 0.88 (t,

3H, J = 7.0 Hz, (CH2)15CH3); 13C NMR (CDCl3) δ 170.9, 169.4, 169.3, 169.1 (C(O)CH3 x 4), 92.2 (C-1), 80.5

(C-3), 73.2 (C-5), 72.8 (OCH2), 71.7 (C-2), 69.2 (C-4), 62.0 (C-6), 32.1, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8,

29.7, 29.6, 29.5, 26.1, 22.8 (OCH2CH2(CH2)13CH3), 30.3 (OCH2CH2(CH2)9CH3), 21.0, 20.9, 20.9, 20.9

(C(O)CH3 x 4), 14.2 (O(CH2)15CH3); LRMS m/z calcd for C30H52O10 [M+Na]+: 595.3. Found 595.3.

1,2,4,6-Tetra-O-acetyl-3-O-(2-methylpropyl)-β-D-glucopyranose (4g)

Compound 4g was obtained as a colorless liquid (1.25 g, 65 %); RF = 0.44 (hexanes/EtOAc, 67:33); 1H NMR

(CDCl3) δ 5.64 (d, 1H, J1,2 = 8.2 Hz, H-1), 5.11 (m, 1H, H-2), 5.09 (m, 1H, H-4), 4.22 (dd, 1H, 2J6a,6b = 12.4 Hz,

J5,6a = 4.9 Hz, H-6a), 4.09 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6b = 2.3 Hz, H-6b), 3.72 (ddd, 1H, J4,5 = 9.9 Hz, J5,6a =

4.5 Hz, J5,6b = 2.4 Hz, H-5), 3.56 (dd, 1H, J2,3 = 9.2 Hz, J3,4 = 9.2 Hz, H-3), 3.30 (dd, 2H, J = 6.3 Hz, 2J = 2.2

Hz, OCH2), 2.10, 2.08, 2.08, 2.07 (s, 12H, C(O)CH3 x 4), 1.74 (nonet, 1H, J = 6.6 Hz, OCH2CH), 0.84 (d, 6H, J

= 6.7 Hz, OCH2CH(CH3)2); 13C NMR (CDCl3) δ 170.7, 169.3, 169.2, 169.0 (C(O)CH3 x 4), 92.0 (C-1), 80.4

(C-3), 79.4 (OCH2), 73.0 (C-5), 71.5 (C-2), 69.1 (C-4), 61.8 (C-6), 28.9 (OCH2CH), 20.9, 20.8, 20.8, 20.8

(C(O)CH3 x 4), 19.1, 19.0 (OCH2CH(CH3)2); LRMS m/z calcd for C18H28O10 [M+Na]+: 427.2. Found 427.2.

S13

1,2,4,6-Tetra-O-acetyl-3-O-(2-ethylbutyl)-β-D-glucopyranose (4h)

Compound 4h was obtained as a white solid (0.98 g, 98 %); RF = 0.47 (hexanes/EtOAc, 67:33); 1H NMR




Hz, J3,4 = 9.3 Hz, H-3), 3.46 (dd, 2H, J = 4.4 Hz, 2J = 2.6 Hz, OCH2), 2.10, 2.08, 2.08, 2.07 (s, 12H, C(O)CH3 x

4), 1.27 (m, 5H, OCH2CH(CH2CH3)2), 0.83 (t, 6H, J = 7.3 Hz, OCH2CH(CH2CH3)2); 13C NMR (CDCl3) δ

170.9, 169.4, 169.3, 169.1 (C(O)CH3 x 4), 92.2 (C-1), 80.4 (C-3), 74.9 (OCH2), 73.2 (C-5), 71.8 (C-2), 69.4 (C-

4), 61.9 (C-6), 42.0 (OCH2CH), 23.1, 23.1 (OCH2CH(CH2CH3)2), 21.0, 20.9, 20.9, 20.8 (C(O)CH3 x 4), 11.2,

11.2 (OCH2CH(CH2CH3)2); LRMS m/z calcd for C20H32O10 [M+Na]+: 455.2. Found 455.2.

General Procedure for Selective Deacetylation and Phosphorylation (5a-h and 6a-h, respectively)

ORO

AcO

OAc

OAcOH

ORO

AcO

OAc

AcO O PO

OPhOPh

DMF DMAP

ClPO(OPh)2

CH2Cl2

hydrazine



acetateORO

AcO

OAc

OAcOAc


To a stirring solution of the acetylated glucopyranose (4a-h) (2.41 mmol) in anhydrous DMF (10 mL) was

added hydrazine acetate (0.33 g, 3.62 mmol) and the solution subsequently heated to 60ºC for 15 min. The

mixture was then stirred for 1 hr at rt before being diluted with H2O (20 mL) and extracted with

dichloromethane (2 x 20 mL). The combined organic layers were washed with brine (10 mL) and dried

(Na2SO4), filtered and concentrated. The major product was purified by flash chromatography to give the

selectively deacetylated pyranose (5a-h). Under a nitrogen atmosphere, a solution of the selectively

S14

deacetylated product (5a-h) (1.49 mmol) and 4-dimethylaminopyridine (438 mg, 3.58 mmol) in anhydrous

dichloromethane (8 mL) was stirred for 15 min at rt. The flask was cooled to -10ºC and charged with diphenyl

chlorophosphate (464 µL, 2.24 mmol). After 1.5 hr, the mixture was diluted with H2O (20 mL) and extracted

with dichloromethane (2 x 15 mL). The combined organic extracts were washed with brine (10 mL) and dried

(Na2SO4), filtered and concentrated. Purification by flash chromatography afforded the title compounds (6a-g).

Diphenyl 2,4,6-tri-O-acetyl-3-O-methyl-α-D-glucopyranosyl-1-phosphate (6a)

Compound 5a3 (RF = 0.28 (hexanes/EtOAc, 60:40)), a colorless liquid, was provided by silica gel

chromatography (hexanes/EtOAc, 60:40) (556 mg, 72%) and immediately carried onto the subsequent

phosphorylation. Chromatographic purification (hexanes/EtOAc, 72:28) furnished compound 6a as a colorless

liquid (552 mg, 67%); RF = 0.36 (hexanes/EtOAc, 67:33); 1H NMR (CDCl3) δ 7.38-7.20 (m, 10H, Ph x 2), 6.06

(dd, 1H, 1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2 = 3.3 Hz, H-1), 5.07 (dd, 1H, J4,5 = 10.2 Hz, J3,4 = 9.6 Hz, H-4), 4.90

(ddd, 1H, J2,3 = 10.0 Hz, J1,2 = 3.1 Hz, 4J1,P = 3.1 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a = 4.3 Hz, H-6a),

4.02 (ddd, 1H, J4,5 = 10.4 Hz, J5,6a = 4.2 Hz, J5,6b = 2.1 Hz, H-5), 3.90 (dd, 1H, J6a,6b = 12.2 Hz, J5,6b = 2.2 Hz,

H-6b), 3.73 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 9.7 Hz, H-3), 3.45 (s, 3H, CH3), 2.10, 2.00, 1.94 (s, 9H, C(O)CH3 x 3);

13C NMR (CDCl3) δ 170.8, 170.0, 169.4, (C(O)CH3 x 3), 150.6-120.1 (12C, Ph x 2), 95.6 (d, 2J1,P = 6.3 Hz, C-

1), 77.9 (C-3), 71.9 (d, 3J1,P = 7.1 Hz, C-2), 70.3 (C-5), 68.7 (C-4), 61.5 (C-6), 60.6 (CH3), 20.9, 20.8, 20.7

(C(O)CH3 x 3); 31P NMR (CDCl3) δ -13.98 (s, 1P, P-1); LRMS m/z calcd for C25H29O12P [M+Na]+: 575.1.

Found 575.0.

Diphenyl 2,4,6-tri-O-acetyl-3-O-butyl-α-D-glucopyranosyl-1-phosphate (6b)

Compound 5b (RF = 0.43 (hexanes/EtOAc, 67:33)), a colorless liquid, was provided by silica gel


phosphorylation. Chromatographic purification (hexanes/EtOAc, 78:22) furnished compound 6b as a colorless 3 Han, X. B.; Jiang, H.; Schmidt, R. R. Liebigs Ann. Chem. 1993, 8, 853-8.

S15


(dd, 1H, 1JC,H = 183 Hz, J1,P = 6.6 Hz, J1,2 = 3.3 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4),

4.90 (ddd, 1H, J2,3 = 10.0 Hz, J1,2 = 3.2 Hz, 4J1,P = 3.2 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a = 4.2 Hz,

H-6a), 4.01 (ddd, 1H, J4,5 = 10.3 Hz, J5,6a = 4.1 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.5 Hz, J5,6b =

2.2 Hz, H-6b), 3.79 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.56 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s, 9H,

C(O)CH3 x 3), 1.46 (m, 2H, OCH2CH2), 1.30 (m, 2H, OCH2CH2CH2), 0.89 (t, 3H, J = 7.5 Hz, (CH2)3CH3); 13C

NMR (CDCl3) δ 170.7, 169.9, 169.3, (C(O)CH3 x 3), 150.5-120.1 (12C, Ph x 2), 95.7 (d, 2J1,P = 6.0 Hz, C-1),

76.5 (C-3), 73.0 (OCH2), 72.1 (d, 3J1,P = 7.1 Hz, C-2), 70.3 (C-5), 68.9 (C-4), 61.5 (C-6), 32.3 (OCH2CH2),

20.8, 20.7, 20.6 (C(O)CH3 x 3), 19.1 (OCH2CH2CH2), 13.9 ((CH2)3CH3); 31P NMR (CDCl3) δ -14.00 (s, 1P, P-

1); LRMS m/z calcd for C28H35O12P [M+Na]+: 617.2. Found 617.2.

Diphenyl 2,4,6-tri-O-acetyl-3-O-hexyl-α-D-glucopyranosyl-1-phosphate (6c)

Diethyl ether was used instead of dichloromethane as an extraction solvent en route to compound 5c (RF = 0.30

(hexanes/EtOAc, 70:30)), a colorless liquid, which was provided by silica gel chromatography (hexanes/EtOAc,

63:37) (772 mg, 82%) and immediately carried onto the subsequent phosphorylation. Diethyl ether was again

used instead of dichloromethane as an extraction solvent for compound 6c, a colorless liquid, which was

isolated by chromatographic purification (hexanes/EtOAc, 75:25) (538 mg, 58%); RF = 0.29 (hexanes/EtOAc,

75:25); 1H NMR (CDCl3) δ 7.38-7.20 (m, 10H, Ph x 2), 6.06 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2 = 3.4

Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4), 4.90 (ddd, 1H, J2,3 = 9.9 Hz, J1,2 = 3.0 Hz, 4J1,P =

3.0 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.4 Hz, J5,6a = 4.1 Hz,

J5,6b = 2.0 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.6 Hz, J5,6b = 2.1 Hz, H-6b), 3.79 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 9.7

Hz, H-3), 3.55 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 x 3), 1.47 (m, 2H, OCH2CH2), 1.28 (m, 6H,

OCH2CH2(CH2)3CH3), 0.88 (t, 3H, J = 6.8 Hz, O(CH2)5CH3); 13C NMR (CDCl3) δ 170.8, 169.9, 169.3,

(C(O)CH3 x 3), 150.5-120.1 (12C, Ph x 2), 95.7 (d, 2J1,P = 6.0 Hz, C-1), 76.5 (C-3), 73.4 (OCH2), 72.1 (d, 3J1,P

= 7.3 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5 (C-6), 31.7, 25.8, 22.7 (OCH2CH2(CH2)3CH3), 30.3 (OCH2CH2),

S16

20.9, 20.8, 20.6 (C(O)CH3 x 3), 14.2 (O(CH2)5CH3); 31P NMR (CDCl3) δ -14.00 (s, 1P, P-1); LRMS m/z calcd

for C30H39O12P [M+Na]+: 645.2. Found 645.1.

Diphenyl 2,4,6-tri-O-acetyl-3-O-octyl-α-D-glucopyranosyl-1-phosphate (6d)

Diethyl ether was used instead of dichloromethane as an extraction solvent en route to compound 5d (RF = 0.27

(hexanes/EtOAc, 67:33)), a colorless liquid which was provided by silica gel chromatography (hexanes/EtOAc,

63:37) (625 mg, 62%) and immediately carried onto the subsequent phosphorylation. Chromatographic

purification (hexanes/EtOAc, 81:19) furnished compound 6d as a colorless liquid (717 mg, 74%); RF = 0.45

(hexanes/EtOAc, 67:33); 1H NMR (CDCl3) δ 7.38-7.20 (m, 10H, Ph x 2), 6.06 (dd, 1H, 1JC,H = 181 Hz, J1,P =

6.6 Hz, J1,2 = 3.4 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.2 Hz, J3,4 = 10.2 Hz, H-4), 4.90 (ddd, 1H, J2,3 = 10.1 Hz, J1,2

= 3.2 Hz, 4J1,P = 3.2 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a = 4.2 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.2

Hz, J5,6a = 3.9 Hz, J5,6b = 2.0 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.6 Hz, J5,6b = 2.1 Hz, H-6b), 3.78 (dd, 1H, J2,3 =

9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.55 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 x 3), 1.47 (m, 2H,

OCH2CH2), 1.34-1.19 (m, 10H, OCH2CH2(CH2)5CH3), 0.88 (t, 3H, J = 7.1 Hz, (CH2)7CH3); 13C NMR (CDCl3)

δ 170.8, 169.9, 169.3, (C(O)CH3 x 3), 150.6-120.1 (12C, Ph x 2), 95.7 (d, 2J1,P = 6.1 Hz, C-1), 76.5 (C-3), 73.5

(OCH2), 72.1 (d, 3J1,P = 7.2 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5 (C-6), 32.0, 29.5, 29.4, 26.1, 22.8

(OCH2CH2(CH2)5CH3), 30.3 (OCH2CH2(CH2)5CH3), 20.9, 20.8, 20.6 (C(O)CH3 x 3), 14.2 ((CH2)7CH3); 31P

NMR (CDCl3) δ -14.00 (s, 1P, P-1); LRMS m/z calcd for C32H43O12P [M+Na]+: 673.2. Found 673.3.

Diphenyl 2,4,6-tri-O-acetyl-3-O-dodecyl-α-D-glucopyranosyl-1-phosphate (6e)

Diethyl ether was used instead of dichloromethane as an extraction solvent en route to compound 5e4 (RF = 0.29

(hexanes/EtOAc, 67:33)), a colorless liquid, which was not purified by silica gel chromatography (675 mg,

59%). For the phosphorylation, diethyl ether was again used instead of dichloromethane as an extraction

solvent, which was isolated by chromatographic purification (hexanes/EtOAc, 80:20) to furnish compound 6e 4 Miethchen, R.; Holz, J.; Prade, H.; Peters, D. Ger. (East) 1991, 3 pp.

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as a colorless liquid (463 mg, 44%); RF = 0.38 (hexanes/EtOAc, 75:25); 1H NMR (CDCl3) δ 7.38-7.20 (m, 10H,

Ph x 2), 6.05 (dd, 1H, 1JC,H = 180 Hz, J1,P = 6.6 Hz, J1,2 = 3.4 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.3 Hz, J3,4 = 9.7

Hz, H-4), 4.90 (ddd, 1H, J2,3 = 10.0 Hz, J1,2 = 3.1 Hz, 4J1,P = 3.1 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.5 Hz, J5,6a =

4.2 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.4 Hz, J5,6a = 4.1 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.5 Hz,

J5,6b = 2.1 Hz, H-6b), 3.79 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.55 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s,

9H, C(O)CH3 x 3), 1.47 (m, 2H, OCH2CH2), 1.33-1.19 (m, 18H, OCH2CH2(CH2)9CH3), 0.88 (t, 3H, J = 6.7 Hz,

O(CH2)11CH3); 13C NMR (CDCl3) δ 170.8, 169.9, 169.3, (C(O)CH3 x 3), 150.6-120.1 (12C, Ph x 2), 95.7 (d,

2J1,P = 6.1 Hz, C-1), 76.6 (C-3), 73.5 (OCH2), 72.1 (d, 3J1,P = 7.3 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5 (C-6),

32.1, 29.8, 29.8, 29.8, 29.8, 29.6, 29.5, 26.1, 22.8 (OCH2CH2(CH2)9CH3), 30.4 (OCH2CH2(CH2)9CH3), 20.9,

20.8, 20.7 (C(O)CH3 x 3), 14.2 (O(CH2)11CH3); 31P NMR (CDCl3) δ -14.00 (s, 1P, P-1); LRMS m/z calcd for

C36H51O12P [M+Na]+: 729.3. Found 729.1.

Diphenyl 2,4,6-tri-O-acetyl-3-O-hexadecyl-α-D-glucopyranosyl-1-phosphate (6f)

Chloroform was used instead of dichloromethane as an extraction solvent en route to compound 5f4 (RF = 0.21

(hexanes/EtOAc, 67:33)), a white solid, which was provided by silica gel chromatography (hexanes/EtOAc,

67:33) (524 mg, 41%) and immediately carried onto the subsequent phosphorylation. Chromatographic

purification (hexanes/EtOAc, 80:20) furnished compound 6f as a colorless liquid (625 mg, 55%); RF = 0.25

(hexanes/EtOAc, 80:20); 1H NMR (CDCl3) δ 7.39-7.18 (m, 10H, Ph x 2), 6.06 (dd, 1H, 1JC,H = 180 Hz, J1,P =

6.6 Hz, J1,2 = 3.3 Hz, H-1), 5.08 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4), 4.90 (ddd, 1H, J2,3 = 10.0 Hz, J1,2

= 3.0 Hz, 4J1,P = 3.0 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.6 Hz, J5,6a = 4.3 Hz, H-6a), 4.01 (ddd, 1H, J4,5 = 10.3

Hz, J5,6a = 3.9 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.5 Hz, J5,6b = 2.2 Hz, H-6b), 3.79 (dd, 1H, J2,3 =

9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.54 (m, 2H, OCH2), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 x 3), 1.47 (m, 2H,

OCH2CH2), 1.33-1.19 (m, 26H, OCH2CH2(CH2)13CH3), 0.88 (t, 3H, J = 7.1 Hz, O(CH2)15CH3); 13C NMR

(CDCl3) δ 170.8, 169.9, 169.3, (C(O)CH3 x 3), 150.6-120.1 (12C, Ph x 2), 95.7 (d, 2J1,P = 6.2 Hz, C-1), 76.5 (C-

3), 73.4 (OCH2), 72.1 (d, 3J1,P = 7.2 Hz, C-2), 70.4 (C-5), 68.9 (C-4), 61.5 (C-6), 32.0, 29.8, 29.8, 29.8, 29.8,

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29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 26.1, 22.8 (OCH2CH2(CH2)13CH3), 30.3 (OCH2CH2(CH2)13CH3), 20.9, 20.7,

20.6 (C(O)CH3 x 3), 14.2 (O(CH2)15CH3); 31P NMR (CDCl3) δ -14.00 (s, 1P, P-1); LRMS m/z calcd for

C40H59O12P [M+Na]+: 785.4. Found 785.4.

Diphenyl 2,4,6-tri-O-acetyl-3-O-(2-methylpropyl)-α-D-glucopyranosyl-1-phosphate (6g)

Compound 5g (RF = 0.23 (hexanes/EtOAc, 67:33)), a colorless liquid, was provided by silica gel


phosphorylation. Chromatographic purification (hexanes/EtOAc, 71:29) furnished compound 6g as a colorless


(dd, 1H, 1JC,H = 180 Hz, J1,P = 6.5 Hz, J1,2 = 3.2 Hz, H-1), 5.10 (dd, 1H, J4,5 = 10.0 Hz, J3,4 = 10.0 Hz, H-4),

4.92 (ddd, 1H, J2,3 = 9.9 Hz, J1,2 = 3.0 Hz, 4J1,P = 3.0 Hz, H-2), 4.14 (dd, 1H, 2J6a,6b = 12.6 Hz, J5,6a = 4.2 Hz, H-

6a), 4.01 (ddd, 1H, J4,5 = 10.4 Hz, J5,6a = 4.2 Hz, J5,6b = 2.3 Hz, H-5), 3.90 (dd, 1H, J6a,6b = 12.5 Hz, J5,6b = 2.2

Hz, H-6b), 3.78 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 3.37 (dd, 1H, J = 6.2 Hz, 2J = 8.4 Hz, OCH2a), 3.28

(dd, 1H, J = 6.4 Hz, 2J = 8.8 Hz, OCH2b), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 x 3), 1.75 (nonet, 1H, J = 6.6 Hz,

OCH2CH), 0.84 (d, 6H, J = 6.6 Hz, OCH2CH(CH3)2); 13C NMR (CDCl3) δ 170.8, 170.0, 169.3, (C(O)CH3 x 3),

150.6-120.2 (12C, Ph x 2), 95.8 (d, 2J1,P = 6.1 Hz, C-1), 80.3 (OCH2), 76.7 (C-3), 72.1 (d, 3J1,P = 7.2 Hz, C-2),

70.4 (C-5), 68.9 (C-4), 61.5 (C-6), 29.0 (OCH2CH), 20.9, 20.8, 20.6 (C(O)CH3 x 3), 19.2, 19.2

(OCH2CH(CH3)2); 31P NMR (CDCl3) δ -14.00 (s, 1P, P-1); LRMS m/z calcd for C28H35O12P [M+Na]+: 617.2.

Found 617.2.

Diphenyl 2,4,6-tri-O-acetyl-3-O-(2-ethylbutyl)-α-D-glucopyranosyl-1-phosphate (6h)

Compound 5h (RF = 0.23 (hexanes/EtOAc, 67:33)), a white solid, was provided by silica gel chromatography

(hexanes/EtOAc, 61:39) (612 mg, 65%) and immediately carried onto the subsequent phosphorylation.

Chromatographic purification (hexanes/EtOAc, 78:22) furnished compound 6h as a colorless liquid (724 mg,

78%); RF = 0.24 (hexanes/EtOAc, 78:22); 1H NMR (CDCl3) δ 7.39-7.17 (m, 10H, Ph x 2), 6.05 (dd, 1H, 1JC,H =

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180 Hz, J1,P = 6.6 Hz, J1,2 = 3.4 Hz, H-1), 5.09 (dd, 1H, J4,5 = 10.2 Hz, J3,4 = 9.5 Hz, H-4), 4.91 (ddd, 1H, J2,3 =

10.0 Hz, J1,2 = 3.2 Hz, 4J1,P = 3.2 Hz, H-2), 4.13 (dd, 1H, 2J6a,6b = 12.6 Hz, J5,6a = 4.4 Hz, H-6a), 4.01 (ddd, 1H,

J4,5 = 10.4 Hz, J5,6a = 4.2 Hz, J5,6b = 2.1 Hz, H-5), 3.89 (dd, 1H, J6a,6b = 12.5 Hz, J5,6b = 2.2 Hz, H-6b), 3.78 (dd,

1H, J2,3 = 9.7 Hz, J3,4 = 9.7 Hz, H-3), 3.52 (dd, 1H, J = 4.6 Hz, 2J = 9.0 Hz, OCH2a), 3.42 (dd, 1H, J = 4.4 Hz, 2J

= 8.8 Hz, OCH2b), 2.08, 2.00, 1.92 (s, 9H, C(O)CH3 x 3), 1.28 (m, 5H, OCH2CH(CH2CH3)2), 0.83 (t, 6H, J =

7.0 Hz, OCH2CH(CH3)2); 13C NMR (CDCl3) δ 170.8, 170.0, 169.3, (C(O)CH3 x 3), 150.5-120.2 (12C, Ph x 2),

95.7 (d, 2J1,P = 6.1 Hz, C-1), 76.6 (C-3), 75.4 (OCH2), 72.3 (d, 3J1,P = 7.3 Hz, C-2), 70.4 (C-5), 69.0 (C-4), 61.5

(C-6), 42.0 (OCH2CH), 23.2, 23.2 (OCH2CH(CH2CH3)2), 20.9, 20.8, 20.6 (C(O)CH3 x 3), 11.3, 11.3

(OCH2CH(CH2CH3)2); 31P NMR (CDCl3) δ -13.99 (s, 1P, P-1); LRMS m/z calcd for C30H39O12P [M+Na]+:

645.2. Found 645.2.

General Procedure for Deprotection and Ion Exchange (7a-h and 8a-h, respectively)

ORO

AcO

OAc

AcO O PO

OPhOPh

ORO

HO

OH

HO O PO

OO

NH

EtEtEt

NH

EtEtEt

1) H2, PtO21:1 EtOAc/EtOH

2) MeOH/H2O/NEt32:2:150°C



ORO

HO

OH

HO O PO

OO NH4

NH4

2) NH4OH

1) Amberlite H+


A solution of phosphorylated pyranoside (0.384 mmol) and platinium (IV) oxide (0.242 mmol) in 1:1

EtOH/EtOAc (4 mL) was shaken under H2 in a Parr apparatus at 54 PSI for 2 h. The reaction mixture was

filtered through Celite and concentrated. The residue was taken up in 2:2:1 MeOH/H2O/NEt3 (10 mL) and

stirred at 50ºC for 36 h. Again, the mixture was concentrated and the residue was partitioned between H2O (10

mL) and EtOAc (10 mL). The aqueous layer containing the triethyl ammonium salt (7a-h) was passed through

Amberlite IR-120 PLUS(H) ion exchange resin. The resulting acidic aqueous fraction was immediately

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adjusted to pH 8 with NH4OH 0.1 M, concentrated to 5 mL, and lyophilized. Titration with NH4OH 0.1 M and

lyophilization was repeated twice more to remove NH4OAc and isolate the ammonium salt (8a-h).

3-O-Methyl-α-D-glucopyranosyl phosphate diammonium salt (8a)5

Deacetylation was performed at rt and compound 8a was isolated as a colorless foam (50 mg, 42%); 1H NMR

(D2O) δ 5.35 (dd, 1H, 1JC,H = 176 Hz, J1,P = 7.2 Hz, J1,2 = 3.0 Hz, H-1), 3.80 (ddd, 1H, J4,5 = 9.6 Hz, J5,6b = 4.7

Hz, J5,6a = 2.0 Hz, H-5), 3.75 (dd, 1H, 2J6a,6b = 12.2 Hz, J5,6a = 2.0 Hz, H-6a), 3.65 (dd, 1H, J6a,6b = 12.4 Hz, J5,6b

= 4.9 Hz, H-6b), 3.52 (s, 3H, CH3), 3.47 (m, 1H, H-2), 3.44 (m, 1H, H-3), 3.39 (m, 1H, H-4); 13C NMR (D2O) δ

94.2 (d, 2J1,P = 5.7 Hz, C-1), 82.8 (C-3), 72.3 (C-5), 71.4 (d, 3J1,P = 7.5 Hz, C-2), 69.0 (C-4), 60.5 (C-6), 60.0

(CH3); 31P NMR (D2O) δ 0.77 (s, 1P, P-1); HRMS m/z calcd for C7H13O9P2- [M+H]-: 273.0386. Found

273.0390.

3-O-Butyl-α-D-glucopyranosyl phosphate diammonium salt (8b)

Compound 8b was obtained as a colorless foam (63 mg, 47%); 1H NMR (D2O) δ 5.33 (dd, 1H, 1JC,H = 172 Hz,

J1,P = 7.4 Hz, J1,2 = 3.4 Hz, H-1), 3.79 (ddd, 1H, J4,5 = 10.1 Hz, J5,6b = 5.0 Hz, J5,6a = 2.2 Hz, H-5), 3.75 (dd, 1H,

2J6a,6b = 12.5 Hz, J5,6a = 2.2 Hz, H-6a), 3.72 (t, 2H, J = 6.7 Hz, OCH2), 3.63 (dd, 1H, J6a,6b = 12.5 Hz, J5,6b = 5.2

Hz, H-6b), 3.50 (dd, 1H, J2,3 = 9.5 Hz, J3,4 = 9.2 Hz, H-3), 3.42 (ddd, 1H, J2,3 = 9.6 Hz, J1,2 = 3.3 Hz, 4J2,P = 2.0

Hz, H-2), 3.33 (dd, 1H, J3,4 = 9.6 Hz, J4,5 = 9.6 Hz, H-4), 1.49 (p, 2H, J = 7.0 Hz, OCH2CH2), 1.26 (sextet, 2H,

J = 7.5 Hz, O(CH2)2CH2CH3), 0.79 (t, 3H, J = 7.8 Hz, O(CH2)3CH3); 13C NMR (D2O) δ 94.1 (d, 2J1,P = 5.5 Hz,

C-1), 81.5 (C-3), 73.1 (OCH2), 72.3 (C-5), 71.8 (d, 3J1,P = 7.4 Hz, C-2), 69.3 (C-4), 60.7 (C-6), 31.5

(OCH2CH2), 18.5 (OCH2CH2CH2), 13.1 (O(CH2)3CH3); 31P NMR (D2O) δ 1.32 (s, 1P, P-1); HRMS m/z calcd

for C10H19O9P2- [M+H]-: 315.0856. Found 315.0856.

3-O-Hexyl-α-D-glucopyranosyl phosphate diammonium salt (8c)

5 Jiang, J.; Albermann, C.; Thorson, J. ChemBioChem. 2003, 4, 443-446.

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Compound 8c was obtained as a colorless foam (110 mg, 76%); 1H NMR (D2O) δ 5.34 (dd, 1H, 1JC,H = 173 Hz,

J1,P = 6.9 Hz, J1,2 = 3.0 Hz, H-1), 3.78 (m, 1H, H-5), 3.74 (m, 1H, H-6a), 3.70 (t, 2H, J = 6.6 Hz, OCH2), 3.64

(dd, 1H, J6a,6b = 12.0 Hz, J5,6b = 4.5 Hz, H-6b), 3.49 (dd, 1H, J2,3 = 9.3 Hz, J3,4 = 9.3 Hz, H-3), 3.44 (ddd, 1H,

J2,3 = 9.7 Hz, H-2), 3.35 (dd, 1H, J3,4 = 9.5 Hz, J4,5 = 9.5 Hz, H-4), 1.50 (p, 2H, J = 7.0 Hz, OCH2CH2), 1.22 (m,

6H, OCH2CH2(CH2)3CH3), 0.76 (m, 3H, O(CH2)5CH3); 13C NMR (D2O) δ 94.4 (d, 2J1,P = 5.7 Hz, C-1), 81.4 (C-

3), 73.4 (OCH2), 72.4 (C-5), 71.6 (d, 3J1,P = 7.7 Hz, C-2), 69.1 (C-4), 60.6 (C-6), 30.9, 24.8, 22.0

(OCH2CH2(CH2)3CH3), 29.2 (OCH2CH2), 13.4 (O(CH2)5CH3); 31P NMR (D2O) δ 0.41 (s, 1P, P-1); HRMS m/z

calcd for C12H23O9P2- [M+H]-: 343.1169. Found 343.1176.

3-O-Octyl-α-D-glucopyranosyl phosphate diammonium salt (8d)

Diethyl ether was used instead of ethyl acetate as an extraction solvent en route to compound 8d, a colorless

foam (66 mg, 42%); 1H NMR (D2O) δ 5.36 (dd, 1H, 1JC,H = 173 Hz, J1,P = 7.2 Hz, J1,2 = 3.2 Hz, H-1), 3.78

(ddd, 1H, J4,5 = 9.8 Hz, J5,6b = 5.0 Hz, J5,6a = 2.1 Hz, H-5), 3.75 (m, 1H, H-6a), 3.72 (t, 2H, J = 7.0 Hz, OCH2),

3.66 (dd, 1H, J6a,6b = 12.3 Hz, J5,6b = 4.9 Hz, H-6b), 3.50 (m, 1H, H-3), 3.45 (m, 1H, H-2), 3.37 (dd, 1H, J3,4 =

9.4 Hz, J4,5 = 9.4 Hz, H-4), 1.50 (p, 2H, J = 7.2 Hz, OCH2CH2), 1.30-1.12 (m, 10H, OCH2CH2(CH2)5CH3), 0.77

(t, 3H, J = 7.0 Hz, (CH2)7CH3); 13C NMR (D2O) δ 94.6 (d, 2J1,P = 5.9 Hz, C-1), 81.4 (C-3), 73.5 (OCH2), 72.5

(C-5), 71.5 (d, 3J1,P = 7.7 Hz, C-2), 69.1 (C-4), 60.5 (C-6), 31.1, 28.5, 28.4, 25.1, 22.0 (OCH2CH2(CH2)5CH3),

29.3 (OCH2CH2), 13.4 (O(CH2)7CH3); 31P NMR (D2O) δ 0.12 (s, 1P, P-1); HRMS m/z calcd for C14H28O9P2-

[M+H]-: 371.1482. Found 371.1490.

3-O-Dodecyl-α-D-glucopyranosyl phosphate diammonium salt (8e)

Deacetylation was performed at 70ºC instead of 50ºC and diethyl ether was used instead of ethyl acetate as an

extraction solvent. Triethylamine (1 mL) was added before rotary evaporation to suppress the surfactant-like

behavior of the salt. After the third NH4OAc sublimation, the residue was dissolved in water and filtered

through a short pad of Bio-Rad Chelex 100. The solution was then passed through Amberlite IR-120 PLUS(H)

ion exchange resin, titrated with NH4OH 0.1 M, and lyophilized to forge compound 8e as a colorless foam (103

S22

mg, 58%); 1H NMR (MeOD) δ 5.48 (dd, 1H, 1JC,H = 173 Hz, J1,P = 6.9 Hz, J1,2 = 3.3 Hz, H-1), 3.85 (ddd, 1H,

J4,5 = 9.8 Hz, J5,6b = 5.7 Hz, J5,6a = 2.1 Hz, H-5), 3.81 (m, 2H, OCH2), 3.80 (m, 1H, H-6a), 3.63 (dd, 1H, J6a,6b =

11.6 Hz, J5,6b = 5.5 Hz, H-6b), 3.47 (dd, 1H, J2,3 = 9.0 Hz, J3,4 = 9.0 Hz, H-3), 3.42 (ddd, 1H, J2,3 = 9.6 Hz, J1,2

= 3.0 Hz, 4J1,P = 3.0 Hz, H-2), 3.32 (m, 1H, H-4), 1.62 (p, 2H, J = 7.3 Hz, OCH2CH2), 1.41-1.23 (m, 18H,

OCH2CH2(CH2)9CH3), 0.90 (t, 3H, J = 7.0 Hz, (CH2)11CH3); 13C NMR (MeOD) δ 96.6 (d, 2J1,P = 6.2 Hz, C-1),

83.6 (C-3), 74.6 (OCH2), 74.5 (C-5), 74.0 (d, 3J1,P = 7.5 Hz, C-2), 71.4 (C-4), 62.8 (C-6), 33.1, 30.8, 30.8, 30.8,

30.8, 30.7, 30.5, 27.1, 23.7 (OCH2CH2(CH2)9CH3), 31.4 (OCH2CH2), 14.4 (O(CH2)11CH3); 31P NMR (MeOD) δ

-1.86 (s, 1P, P-1); HRMS m/z calcd for C18H35O9P2- [M+H]-: 427.2108. Found 427.2115.

3-O-Hexadecyl-α-D-glucopyranosyl phosphate diammonium salt (8f)

Deacetylation was performed at 70ºC instead of 50ºC and diethyl ether was used instead of ethyl acetate as an

extraction solvent. Triethylamine (1 mL) was added before rotary evaporation to suppress the surfactant-like

behavior of the salt. After the third NH4OAc sublimation, the residue was dissolved in water and filtered

through a short pad of Bio-Rad Chelex 100. The solution was then passed through Amberlite IR-120 PLUS(H)

ion exchange resin, titrated with NH4OH 0.1 M, and lyophilized to forge compound 8f as a colorless foam (179

mg, 90%). Coupling constants are omitted from the 1H NMR spectrum because of the compound’s inherent

insolubility in a variety of NMR solvents led to significant line-broadening; 1H NMR (D2O) δ 5.44 (d, 1H, 1JC,H

= 174 Hz, H-1), 3.86 (m, 1H, H-5), 3.86 (m, 1H, H-6a), 3.74 (m, 2H, OCH2), 3.69 (m, 1H, H-6b), 3.56 (m, 1H,

H-2), 3.52 (m, 1H, H-3), 3.39 (m, 1H, H-4), 1.58 (m, 2H, OCH2CH2), 1.42-1.15 (m, 26H,

OCH2CH2(CH2)13CH3), 0.84 (m, 3H, (CH2)15CH3); 13C NMR (D2O) δ 94.9 (d, 2J1,P = 5.1 Hz, C-1), 81.0 (C-3),

72.7 (C-5), 72.2 (OCH2), 71.1 (d, 3J1,P = 7.3 Hz, C-2), 69.4 (C-4), 61.0 (C-6), 32.1, 30.3, 30.3, 30.3, 30.3, 30.3,

30.3, 30.3, 30.1, 30.1, 30.0, 29.7, 26.2, 22.7 (OCH2(CH2)14CH3), 13.8 (O(CH2)15CH3); 31P NMR (D2O) δ -0.57

(s, 1P, P-1); HRMS m/z calcd for C22H43O9P2- [M+H]-: 483.2734. Found 483.2708.

S23

3-O-(2-Methylpropyl)-α-D-glucopyranosyl phosphate diammonium salt (8g)

Compound 8g was obtained as a colorless foam (69 mg, 51%); 1H NMR (D2O) δ 5.29 (dd, 1H, 1JC,H = 171 Hz,

J1,P = 7.3 Hz, J1,2 = 3.3 Hz, H-1), 3.76 (ddd, 1H, J4,5 = 10.0 Hz, J5,6b = 4.8 Hz, J5,6a = 1.8 Hz, H-5), 3.72 (dd, 1H,

2J6a,6b = 12.3 Hz, J5,6a = 1.9 Hz, H-6a), 3.60 (dd, 1H, J6a,6b = 12.1 Hz, J5,6b = 5.2 Hz, H-6b), 3.47 (dd, 1H, J2,3 =

9.8 Hz, J3,4 = 9.4 Hz, H-3), 3.46 (d, 2H, J = 6.1 Hz, OCH2), 3.41 (m, 1H, H-2), 3.31 (dd, 1H, J3,4 = 9.5 Hz, J4,5 =

9.5 Hz, H-4), 1.74 (nonet, 1H, J = 6.5 Hz, OCH2CH), 0.81, 0.81 (d, 3H, J = 6.7 Hz, OCH2CH(CH3)2); 13C NMR

(D2O) δ 94.0 (d, 2J1,P = 5.6 Hz, C-1), 81.7 (C-3), 80.1 (OCH2), 72.2 (C-5), 71.8 (d, 3J1,P = 7.2 Hz, C-2), 69.3 (C-

4), 60.7 (C-6), 28.2 (OCH2CH), 18.6, 18.6 (OCH2CH(CH3)2); 31P NMR (D2O) δ 1.76 (s, 1P, P-1); HRMS m/z

calcd for C10H19O9P2- [M+H]-: 315.0856. Found 315.0869.

3-O-(2-Ethylbutyl)-α-D-glucopyranosyl phosphate diammonium salt (8h)

Compound 8h was obtained as a colorless foam (145 mg, 76%); 1H NMR (D2O) δ 5.32 (dd, 1H, 1JC,H = 172 Hz,

J1,P = 4.6 Hz, J1,2 = 3.0 Hz, H-1), 3.79 (m, 1H, H-5), 3.75 (d, 1H, 2J6a,6b = 12.6 Hz, H-6a), 3.63 (m, 1H, H-6b),

3.61 (m, 2H, OCH2), 3.49 (dd, 1H, J2,3 = 9.2 Hz, J3,4 = 9.2 Hz, H-3), 3.42 (d, 1H, J2,3 = 9.6 Hz, H-2), 3.33 (dd,

1H, J3,4 = 9.2 Hz, J4,5 = 9.2 Hz, H-4), 1.39 (m, 1H, OCH2CH(CH2CH3)2), 1.25 (m, 4H, OCH2CH(CH2CH3)2),

0.76 (t, 6H, J = 6.7 Hz, OCH2CH(CH2CH3)2); 13C NMR (D2O) δ 94.1 (d, 2J1,P = 5.6 Hz, C-1), 81.7 (C-3), 75.7

(OCH2), 72.3 (C-5), 71.8 (d, 3J1,P = 7.3 Hz, C-2), 69.3 (C-4), 60.7 (C-6), 41.0 (OCH2CH), 22.5, 22.4

(OCH2CH(CH2CH3)2), 10.2, 10.2 (OCH2CH(CH2CH3)2); 31P NMR (D2O) δ 1.62 (s, 1P, P-1); HRMS m/z calcd

for C12H23O9P2- [M+H]-: 343.1169. Found 343.1159.

1H, 13C and 31P NMR Spectra of compounds 6a-h and 8a-h

MH_072x1d_1H CDCl3 {C:\nmr_users} huestis 2

Current Data ParametersNAME MH_072xEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070127Time 13.58INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT CDCl3NS 32DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 287.4DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 9.80 usecPL1 -2.00 dBSFO1 500.1332698 MHz

F2 - Processing parametersSI 65536SF 500.1300374 MHzWDW noSSB 0LB 0.00 HzGB 0PC 1.00

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.000

1.571

1.939

2.003

2.097

3.451

3.712

3.732

3.751

3.887

3.891

3.912

3.916

4.123

4.132

4.148

4.157

4.889

4.895

4.901

4.909

4.915

4.921

5.054

5.074

5.094

6.051

6.057

6.064

6.071

7.204

7.219

7.233

7.243

7.245

7.246

7.253

7.261

7.344

7.347

7.361

7.370

7.375

7.378

2.92

2.93

3.05

2.98

1.00

0.99

1.00

1.00

1.00

1.00

0.97

10.60

4.04.24.44.64.85.0 ppm

3.712

3.732

3.751

3.887

3.891

3.912

3.916

4.123

4.132

4.148

4.157

4.889

4.895

4.901

4.909

4.915

5.054

5.074

5.094

0.996

0.992

0.999

1.000

1.000

1.001

OMeO

AcO

OAc

AcO O PO

OPhOPh

6a

S25

MH_072x1d_13C{1H}_wide CDCl3 {C:\nmr_users} huestis 2


F2 - Acquisition ParametersDate_ 20070127Time 14.07INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 2SWH 33783.785 HzFIDRES 0.515500 HzAQ 0.9699976 secRG 645.1DW 14.800 usecDE 6.00 usecTE 300.4 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 4

======== CHANNEL f1 ========NUC1 13CP1 7.90 usecPL1 0.00 dBSFO1 125.7716270 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -2.00 dBPL12 15.72 dBPL13 19.37 dBSFO2 500.1322696 MHz


30405060708090100110120130140150160170 ppm

20.681

20.764

20.890

60.557

61.487

68.682

70.321

71.863

71.921

76.907

77.161

77.269

77.415

77.859

95.594

95.642

120.150

120.189

120.199

120.334

120.373

125.817

125.827

125.862

125.873

129.965

129.971

130.077

130.090

150.446

150.506

150.565

169.374

170.012

OMeO

AcO

OAc

AcO O PO

OPhOPh

6a

S26

-35-30-25-20-15-10-50 ppm

-13.98


F2 - Acquisition ParametersDate_ 20070127Time 14.59INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 65536SOLVENT CDCl3NS 128DS 0SWH 98039.219 HzFIDRES 1.495960 HzAQ 0.3342887 secRG 8192DW 5.100 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 1

======== CHANNEL f1 ========NUC1 31PP1 8.41 usecPL1 0.50 dBSFO1 202.4664658 MHz



MH_072x1d_31P{1H} CDCl3 {C:\nmr_users} huestis 2

OMeO

AcO

OAc

AcO O PO

OPhOPh

6a

S27

MH_103_clean1d_1H CDCl3 {C:\nmr_users} huestis 14

Current Data ParametersNAME MH_103_cleanEXPNO 10PROCNO 1




7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.873

0.888

0.902

1.299

1.313

1.445

1.458

1.921

2.000

2.080

3.526

3.594

3.786

3.805

3.885

3.906

3.910

4.127

4.135

4.152

4.891

4.911

5.062

5.081

5.101

6.046

6.052

6.059

7.202

7.216

7.231

7.243

7.245

7.247

7.253

7.260

7.263

7.264

7.270

7.340

7.345

7.358

7.372

7.376

3.06

2.15

2.05

3.00

3.01

3.04

1.02

1.02

1.02

1.02

1.02

1.02

1.02

1.01

1.00

10.03

3.84.04.24.44.64.85.0 ppm

3.594

3.612

3.767

3.786

3.805

3.881

3.885

3.906

3.910

4.127

4.135

4.152

4.160

4.891

4.911

5.062

5.081

1.021

1.020

1.020

1.024

1.022

1.016

1.019

1.015

OH3C(H2C)3O

AcO

OAc

AcO O PO

OPhOPh

6b

S28

MH_103_clean1d_13C{1H}_wide CDCl3 {C:\nmr_users} huestis 14






2030405060708090100110120130140150160170 ppm

13.884

19.122

20.586

20.716

20.831

32.310

61.467

68.886

70.348

72.030

72.088

73.021

76.516

76.909

77.164

77.418

95.662

95.711

120.105

120.145

120.292

120.331

125.758

125.769

125.807

125.816

129.908

129.913

130.023

150.411

150.471

150.530

169.258

169.915

OH3C(H2C)3O

AcO

OAc

AcO O PO

OPhOPh

6b

S29

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

-14.00






MH_103_clean1d_31P{1H} CDCl3 {C:\nmr_users} huestis 14

OH3C(H2C)3O

AcO

OAc

AcO O PO

OPhOPh

6b

S30

Gaia-Phosphorylated Hexyl1d_1H CDCl3 {C:\nmr_users} huestis 11

Current Data ParametersNAME Gaia-Phosphorylated HexylEXPNO 10PROCNO 1




7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.868

0.882

0.896

1.255

1.261

1.270

1.272

1.282

1.296

1.471

1.626

1.922

2.001

2.080

3.512

3.585

3.767

3.787

3.806

3.904

3.909

4.125

4.134

4.891

4.911

5.062

5.081

5.101

6.052

6.058

7.217

7.231

7.241

7.243

7.245

7.258

7.261

7.264

7.340

7.345

7.356

7.359

7.372

7.376

3.27

6.65

2.23

3.07

3.01

3.51

2.24

1.03

1.03

1.04

1.05

1.03

1.13

1.00

10.21

3.84.04.24.44.64.85.0 ppm

3.494

3.499

3.512

3.585

3.598

3.603

3.767

3.787

3.806

3.879

3.884

3.904

3.909

4.003

4.125

4.134

4.151

4.159

4.891

4.897

4.911

5.062

5.081

5.101

2.238

1.029

1.026

1.037

1.047

1.030

1.127

OH3C(H2C)5O

AcO

OAc

AcO O PO

OPhOPh

6c

S31

Gaia-Phosphorylated Hexyl1d_13C{1H}_wide CDCl3 {C:\nmr_users} huestis 11






30405060708090100110120130140150160170 ppm

20.751

20.879

22.729

25.776

30.280

31.743

61.487

68.879

70.372

72.049

72.107

73.445

76.545

95.693

95.742

120.132

120.172

120.320

120.358

125.781

125.791

125.834

125.843

129.935

129.941

130.050

130.055

150.434

150.492

169.269

169.945

OH3C(H2C)5O

AcO

OAc

AcO O PO

OPhOPh

6c

S32

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

-14.00






Gaia-Phosphorylated Hexyl1d_31P{1H} CDCl3 {C:\nmr_users} huestis 11

OH3C(H2C)5O

AcO

OAc

AcO O PO

OPhOPh

6c

S33

MH_1151d_1H CDCl3 {C:\nmr_users} huestis 14

Current Data ParametersNAME MH_115EXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070413Time 10.27INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT CDCl3NS 32DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 128DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1



7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.865

0.879

0.892

1.256

1.295

1.457

1.469

1.922

2.001

2.079

3.510

3.582

3.786

3.805

3.879

3.883

3.904

3.909

4.125

4.134

4.890

4.910

5.061

5.080

5.101

6.046

6.052

6.059

7.201

7.215

7.230

7.241

7.243

7.245

7.258

7.261

7.263

7.339

7.344

7.355

7.357

7.367

7.371

7.376

3.08

10.41

2.06

2.99

3.00

3.10

1.05

1.03

1.03

1.00

1.01

1.03

1.02

1.02

1.00

10.58

3.84.04.24.44.64.85.0 ppm

3.601

3.766

3.786

3.805

3.879

3.883

3.904

3.909

4.003

4.020

4.023

4.125

4.134

4.150

4.159

4.884

4.890

4.910

5.061

5.080

1.050

1.032

1.026

1.003

1.011

1.029

1.020

1.016

OH3C(H2C)7O

AcO

OAc

AcO O PO

OPhOPh

6d

S34

MH_1151d_13C{1H}_wide CDCl3 {C:\nmr_users} huestis 14






30405060708090100110120130140150160170 ppm

20.754

20.884

22.765

26.121

29.381

29.534

30.330

31.954

61.492

68.883

70.376

72.055

72.113

73.460

76.547

95.696

95.744

120.135

120.175

120.324

120.363

125.782

125.791

125.834

125.844

129.937

130.055

150.439

150.497

150.504

150.562

169.264

169.941

OH3C(H2C)7O

AcO

OAc

AcO O PO

OPhOPh

6d

S35

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

-14.00






MH_1151d_31P{1H} CDCl3 {C:\nmr_users} huestis 14

OH3C(H2C)7O

AcO

OAc

AcO O PO

OPhOPh

6d

S36






7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.865

0.879

0.893

1.252

1.283

1.573

1.921

2.001

2.079

3.508

3.581

3.785

3.805

3.904

3.908

4.124

4.133

4.889

4.909

5.060

5.079

5.080

5.100

7.215

7.217

7.230

7.232

7.233

7.239

7.242

7.244

7.246

7.255

7.257

7.261

7.339

7.344

7.348

7.354

7.355

7.357

7.359

7.371

7.376

3.32

20.22

2.30

2.96

2.99

3.52

2.36

1.02

1.01

1.03

1.03

1.02

1.12

1.00

10.63

3.84.04.24.44.64.85.0 ppm

3.508

3.581

3.766

3.785

3.805

3.883

3.904

3.908

4.124

4.133

4.149

4.889

4.909

5.060

5.079

2.355

1.016

1.007

1.031

1.035

1.016

1.118

OH3C(H2C)11O

AcO

OAc

AcO O PO

OPhOPh

6e

S37







2030405060708090100110120130140150160170 ppm

20.904

22.827

26.147

29.484

29.613

29.760

29.771

29.780

29.805

30.354

32.056

61.508

68.895

70.392

72.072

72.129

73.481

76.561

95.714

95.763

120.152

120.191

120.341

120.380

125.797

125.807

125.851

125.860

129.952

129.958

130.072

150.520

169.282

169.963

170.807

OH3C(H2C)11O

AcO

OAc

AcO O PO

OPhOPh

6e

S38

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

-14.00







OH3C(H2C)11O

AcO

OAc

AcO O PO

OPhOPh

6e

S39

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.865

0.879

0.893

1.252

1.284

1.468

1.657

1.921

2.000

2.078

3.509

3.581

3.766

3.785

3.805

3.905

3.909

4.126

4.134

4.889

4.909

5.061

5.080

5.100

6.046

6.052

6.059

7.215

7.216

7.230

7.241

7.243

7.245

7.258

7.261

7.263

7.265

7.338

7.343

7.348

7.354

7.358

7.370

7.375

3.14

27.4

82.

15

3.01

2.96

3.08

2.09

1.02

1.01

1.02

1.03

1.02

1.04

1.00

10.1

8

Current Data ParametersNAME Gaia-hexadecyl phos_2EXPNO 10PROCNO 1




Gaia-hexadecyl phos_21d_1H CDCl3 {C:\nmr_users} huestis 27

3.84.04.24.44.64.85.0 ppm

3.490

3.496

3.509

3.581

3.594

3.600

3.766

3.785

3.805

3.880

3.884

3.905

3.909

4.002

4.023

4.126

4.134

4.151

4.159

4.883

4.889

4.896

4.903

4.909

4.916

5.061

5.080

5.100

2.085

1.024

1.009

1.024

1.026

1.018

1.036

OH3C(H2C)15O

AcO

OAc

AcO O PO

OPhOPh

6f

S40

30405060708090100110120130140150160170 ppm

20.74

20.88

22.81

26.12

29.47

29.59

29.74

29.75

29.76

29.78

29.79

29.80

29.81

30.33

32.04

61.48

68.87

70.37

72.05

72.10

73.45

76.54

95.69

95.74

120.13

120.17

120.32

120.36

125.77

125.78

125.83

125.84

129.93

130.05

150.43

150.49

150.55

169.25

169.93

170.77






Gaia-hexadecyl phos_21d_13C{1H}_d CDCl3 {C:\nmr_users} huestis 27

OH3C(H2C)15O

AcO

OAc

AcO O PO

OPhOPh

6f

S41

-50-45-40-35-30-25-20-15-10-525 20 15 10 5 0 ppm

-14.00






Gaia-hexadecyl phos_21d_31P{1H}_d CDCl3 {C:\nmr_users} huestis 27

OH3C(H2C)15O

AcO

OAc

AcO O PO

OPhOPh

6f

S42






7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.865

0.879

0.893

1.252

1.283

1.573

1.921

2.001

2.079

3.508

3.581

3.785

3.805

3.904

3.908

4.124

4.133

4.889

4.909

5.060

5.079

5.080

5.100

7.215

7.217

7.230

7.232

7.233

7.239

7.242

7.244

7.246

7.255

7.257

7.261

7.339

7.344

7.348

7.354

7.355

7.357

7.359

7.371

7.376

3.32

20.22

2.30

2.96

2.99

3.52

2.36

1.02

1.01

1.03

1.03

1.02

1.12

1.00

10.63

4.04.24.44.64.85.0 ppm

3.805

3.883

3.904

3.908

4.124

4.133

4.149

4.889

4.909

5.060

5.079

5.080

1.016

1.007

1.031

1.035

1.016

1.118

O(H3C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6g

S43







30405060708090100110120130140150160170 ppm

20.649

20.775

20.910

29.007

61.501

68.929

70.409

72.070

72.129

76.686

80.252

95.735

95.783

120.153

120.193

120.348

120.386

125.798

125.808

125.850

125.859

129.950

129.956

130.069

150.453

150.575

169.304

169.961

170.813

O(H3C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6g

S44

-30-25-20-15-10-55 0 ppm

-14.00







O(H3C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6g

S45

MH_129b1d_1H CDCl3 {C:\nmr_users} huestis 17

Current Data ParametersNAME MH_129bEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070511Time 5.44INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT CDCl3NS 32DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 362DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1



7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.820

0.833

0.835

0.848

1.256

1.271

1.285

1.296

1.568

1.915

2.005

2.075

3.777

3.796

3.903

3.907

4.117

4.896

4.916

5.074

5.093

5.094

5.114

7.216

7.218

7.219

7.231

7.232

7.234

7.242

7.244

7.245

7.261

7.340

7.345

7.349

7.350

7.355

7.356

7.357

7.359

7.360

7.372

7.377

6.32

5.46

3.13

3.08

3.17

2.11

1.04

1.04

1.05

1.05

1.03

1.04

1.00

10.86

3.63.84.04.24.44.64.85.0 ppm

3.777

3.796

3.903

3.907

4.117

4.126

4.896

4.916

5.074

5.093

5.094

2.107

1.044

1.043

1.049

1.045

1.033

1.044

O(H3CH2C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6h

S46

MH_129b1d_13C{1H}_wide CDCl3 {C:\nmr_users} huestis 17






2030405060708090100110120130140150160170 ppm

11.277

11.327

20.647

20.784

20.918

23.207

23.246

42.009

61.504

69.003

70.414

72.239

72.297

75.392

76.563

95.711

95.760

120.163

120.203

120.359

120.397

125.803

125.812

125.853

125.864

129.959

130.073

150.457

150.514

169.303

169.997

O(H3CH2C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6h

S47

-30-25-20-15-10-55 0 ppm

-13.99






MH_129b1d_31P{1H} CDCl3 {C:\nmr_users} huestis 17

O(H3CH2C)2HCH2CO

AcO

OAc

AcO O PO

OPhOPh

6h

S48

3.43.63.84.04.24.44.64.85.05.25.4 ppm

3.423

3.442

3.460

3.467

3.524

3.633

3.643

3.658

3.667

3.735

3.738

3.759

3.763

3.780

3.784

3.790

3.793

3.799

3.803

3.809

5.340

5.346

5.354

5.360

1.19

0.48

1.03

0.23

2.84

1.15

1.11

1.03

1.00

Current Data ParametersNAME Clean NH4 OMe SaltEXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070330Time 7.37INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgcpgpprTD 8192SOLVENT D2ONS 32DS 2SWH 7507.507 HzFIDRES 0.916444 HzAQ 0.5457038 secRG 161.3DW 66.600 usecDE 6.00 usecTE 305.0 KD1 2.00000000 secd12 0.00002000 secD16 0.00020000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 11.10 usecPL1 -1.40 dBPL9 51.67 dBSFO1 500.1323692 MHz

====== GRADIENT CHANNEL =====GPNAM1 sine.100GPZ1 50.00 %P16 1000.00 usec

F2 - Processing parametersSI 32768SF 500.1300390 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

Clean NH4 OMe Salt1d_water D2O {C:\nmr_users} huestis 12

3.53.63.73.8 ppm

3.388

3.407

3.423

3.442

3.460

3.467

3.524

3.633

3.643

3.658

3.667

3.735

3.738

3.759

3.763

3.780

3.784

3.790

3.793

3.799

3.803

3.809

1.191

0.482

1.025

0.234

2.845

1.147

1.107

1.026

OMeOHO

OH

HO O PO

OO NH4

NH48a

S49

Clean NH4 OMe Salt1d_13C{1H}_wide D2O {C:\nmr_users} huestis 12


F2 - Acquisition ParametersDate_ 20070330Time 7.47INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 65536SOLVENT D2ONS 1024DS 2SWH 33783.785 HzFIDRES 0.515500 HzAQ 0.9699976 secRG 912.3DW 14.800 usecDE 6.00 usecTE 300.6 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 4




6065707580859095 ppm59.992

60.499

69.018

71.399

71.459

72.288

82.810

94.212

94.257

OMeOHO

OH

HO O PO

OO NH4

NH48a

S50

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

0.77






Clean NH4 OMe Salt1d_31P{1H} D2O {C:\nmr_users} huestis 12

OMeOHO

OH

HO O PO

OO NH4

NH48a

S51

1.01.52.02.53.03.54.04.55.05.5 ppm

0.780

0.795

0.810

1.241

1.256

1.271

1.286

1.473

1.489

1.503

3.315

3.334

3.353

3.427

3.433

3.438

3.486

3.505

3.524

3.615

3.625

3.639

3.650

3.702

3.715

3.731

3.737

3.757

3.761

3.775

3.779

3.785

5.322

5.328

5.336

5.343

2.77

2.07

2.28

1.05

1.13

1.16

1.28

2.58

1.09

1.15

1.00


F2 - Acquisition ParametersDate_ 20070405Time 11.23INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgcpgpprTD 8192SOLVENT D2ONS 32DS 2SWH 7507.507 HzFIDRES 0.916444 HzAQ 0.5457038 secRG 128DW 66.600 usecDE 6.00 usecTE 305.0 KD1 2.00000000 secd12 0.00002000 secD16 0.00020000 secTD0 1




MH_1041d_water D2O {C:\nmr_users} huestis 2

3.43.53.63.73.8 ppm

3.315

3.334

3.353

3.427

3.433

3.438

3.486

3.505

3.524

3.615

3.625

3.639

3.650

3.702

3.715

3.731

3.737

3.757

3.761

3.775

3.779

1.054

1.133

1.163

1.276

2.581

1.095

1.151

OH3C(H2C)3O

HO

OH

HO O PO

OO NH4

NH48b

S52

MH_1041d_13C{1H}_wide D2O {C:\nmr_users} huestis 2


F2 - Acquisition ParametersDate_ 20070405Time 11.32INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 65536SOLVENT D2ONS 1024DS 2SWH 33783.785 HzFIDRES 0.515500 HzAQ 0.9699976 secRG 1024DW 14.800 usecDE 6.00 usecTE 300.6 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 4




1520253035404550556065707580859095 ppm13.129

18.536

31.462

60.652

69.281

71.724

71.782

72.281

73.059

81.525

94.109

94.154

OH3C(H2C)3O

HO

OH

HO O PO

OO NH4

NH48b

S53

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

1.32






MH_1041d_31P{1H} D2O {C:\nmr_users} huestis 2

OH3C(H2C)3O

HO

OH

HO O PO

OO NH4

NH48b

S54

1.01.52.02.53.03.54.04.55.05.5 ppm

0.760

1.189

1.251

1.489

1.504

1.517

3.330

3.349

3.368

3.429

3.447

3.475

3.493

3.512

3.624

3.633

3.649

3.658

3.694

3.708

3.726

3.756

3.777

4.656

5.337

5.344

5.351

2.86

7.14

2.62

1.17

1.18

0.86

1.44

4.35

0.93

1.00

Current Data ParametersNAME hexyl_NH4EXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070618Time 21.23INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgcpgpprTD 8192SOLVENT D2ONS 32DS 2SWH 7507.507 HzFIDRES 0.916444 HzAQ 0.5457038 secRG 90.5DW 66.600 usecDE 6.00 usecTE 305.0 KD1 2.00000000 secd12 0.00002000 secD16 0.00020000 secTD0 1




hexyl_NH41d_water D2O {C:\nmr_users} huestis 2

3.53.63.73.8 ppm

3.349

3.368

3.429

3.447

3.475

3.493

3.633

3.649

3.658

3.694

3.708

3.726

3.756

3.777

1.165

1.180

0.861

1.436

4.346

0.934

OH3C(H2C)5O

HO

OH

HO O PO

OO NH4

NH48c

S55

2030405060708090100 ppm13.35

21.95

24.83

29.27

30.88

60.55

69.16

71.55

71.61

72.42

73.44

81.42

94.42

94.47






hexyl_NH41d_13C{1H}_n D2O {C:\nmr_users} huestis 2

OH3C(H2C)5O

HO

OH

HO O PO

OO NH4

NH48c

S56

-70-60-50-40-30-20-1040 30 20 10 0 ppm

0.41






hexyl_NH41d_31P{1H}_d D2O {C:\nmr_users} huestis 2

OH3C(H2C)5O

HO

OH

HO O PO

OO NH4

NH48c

S57

1.01.52.02.53.03.54.04.55.05.5 ppm

0.753

0.767

0.775

0.780

1.183

1.194

1.201

1.246

1.260

1.507

1.521

1.535

3.368

3.387

3.465

3.470

3.487

3.505

3.652

3.667

3.676

3.707

3.721

3.736

3.741

3.767

5.350

5.356

5.364

5.370

2.44

10.9

0

2.38

1.04

2.21

1.27

3.10

1.58

1.00


F2 - Acquisition ParametersDate_ 20070420Time 14.04INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgcpgpprTD 8192SOLVENT D2ONS 32DS 2SWH 7507.507 HzFIDRES 0.916444 HzAQ 0.5457038 secRG 128DW 66.600 usecDE 6.00 usecTE 305.0 KD1 2.00000000 secd12 0.00002000 secD16 0.00020000 secTD0 1




MH_1161d_water D2O {C:\nmr_users} huestis 7

3.53.63.73.8 ppm

3.368

3.387

3.465

3.470

3.487

3.505

3.652

3.667

3.676

3.707

3.721

3.736

3.741

1.043

2.215

1.267

3.096

1.575

OH3C(H2C)7O

HO

OH

HO O PO

OO NH4

NH48d

S58







1520253035404550556065707580859095 ppm13.427

22.033

25.155

28.427

28.527

29.303

31.134

60.519

69.136

71.518

71.580

72.467

73.486

81.400

94.530

94.578

OH3C(H2C)7O

HO

OH

HO O PO

OO NH4

NH48d

S59

-80-70-60-50-40-30-20-1060 50 40 30 20 10 0 ppm

0.12







OH3C(H2C)7O

HO

OH

HO O PO

OO NH4

NH48d

S60

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.882

0.896

0.901

0.909

1.334

1.355

1.372

1.385

1.605

1.619

1.634

3.307

3.311

3.314

3.334

3.406

3.420

3.426

3.432

3.455

3.473

3.491

3.614

3.626

3.638

3.649

3.765

3.769

3.783

3.797

3.811

3.824

3.830

3.835

3.840

3.844

3.850

3.854

3.858

3.865

5.471

5.478

5.485

5.492

2.67

20.6

3

2.45

3.44

1.17

1.21

1.32

0.97

1.23

1.22

0.98

1.02

Current Data ParametersNAME MH_137_NH4EXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070618Time 15.53INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgcpgpprTD 8192SOLVENT MeODNS 32DS 2SWH 7507.507 HzFIDRES 0.916444 HzAQ 0.5457038 secRG 181DW 66.600 usecDE 6.00 usecTE 305.0 KD1 2.00000000 secd12 0.00002000 secD16 0.00020000 secTD0 1




MH_137_NH41d_water MeOD {C:\nmr_users} huestis 1

3.43.53.63.73.8 ppm

3.307

3.311

3.314

3.334

3.420

3.426

3.432

3.455

3.473

3.491

3.614

3.626

3.638

3.649

3.765

3.769

3.783

3.797

3.811

3.824

3.830

3.835

3.840

3.844

3.850

3.854

3.444

1.169

1.211

1.319

0.966

1.232

1.217

0.984

OH3C(H2C)11O

HO

OH

HO O PO

OO NH4

NH48e

S61

100 90 80 70 60 50 40 30 20 10 ppm

14.41

23.72

27.15

30.46

30.71

30.75

30.77

30.79

31.36

33.06

48.47

48.48

48.50

48.53

48.55

48.61

48.64

48.65

48.67

48.68

48.71

48.74

48.76

48.79

48.81

48.86

48.88

48.89

48.92

48.94

48.95

48.97

48.98

49.04

49.07

49.09

49.10

49.11

49.12

49.13

49.15

49.20

49.22

49.25

49.28

49.29

49.30

49.31

49.32

49.33

49.35

49.38

49.45

49.46

49.49

49.52

49.85

62.80

71.40

74.01

74.06

74.49

74.55

83.56

96.61

96.66


F2 - Acquisition ParametersDate_ 20070618Time 17.50INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 54048SOLVENT MeODNS 1024DS 4SWH 33783.785 HzFIDRES 0.625070 HzAQ 0.7999752 secRG 9195.2DW 14.800 usecDE 21.14 usecTE 300.4 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 4




MH_137_NH41d_13C{1H}_n MeOD {C:\nmr_users} huestis 1

OH3C(H2C)11O

HO

OH

HO O PO

OO NH4

NH48e

S62

-35-30-25-20-15-10-520 15 10 5 0 ppm

-1.86


F2 - Acquisition ParametersDate_ 20070618Time 16.01INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zgpg30TD 65536SOLVENT MeODNS 512DS 4SWH 98039.219 HzFIDRES 1.495960 HzAQ 0.3342887 secRG 9195.2DW 5.100 usecDE 7.29 usecTE 300.4 KD1 1.00000000 secd11 0.03000000 secDELTA 0.89999998 secTD0 2




MH_137_NH41d_31P{1H}_d MeOD {C:\nmr_users} huestis 1

OH3C(H2C)11O

HO

OH

HO O PO

OO NH4

NH48e

S63

1.01.52.02.53.03.54.04.55.05.5 ppm

0.842

1.165

1.180

1.188

1.391

1.397

1.401

1.581

3.373

3.390

3.408

3.506

3.523

3.554

3.574

3.677

3.678

3.690

3.691

3.700

3.701

3.740

3.852

3.873

5.444

5.450

3.27

27.7

9

2.12

1.09

2.14

3.11

2.14

1.00

Current Data ParametersNAME Hexadecyl deprotected 3EXPNO 11PROCNO 1

F2 - Acquisition ParametersDate_ 20070719Time 2.00INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT D2ONS 128DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 228.1DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1



Gaia-4,6-benzylidene1d_1H D2O {C:\nmr_users} huestis 4

3.53.63.73.83.9 ppm

3.373

3.390

3.408

3.506

3.523

3.554

3.574

3.677

3.678

3.690

3.691

3.700

3.701

3.740

3.852

3.873

1.092

2.143

3.112

2.135

OH3C(H2C)15O

HO

OH

HO O PO

OO NH4

NH48f

S64

2030405060708090 ppm13.83

13.87

22.73

26.15

29.71

29.77

29.96

29.99

30.09

30.27

32.12

61.07

61.09

69.37

71.11

71.12

72.27

72.72

81.03

94.84

94.89

Current Data ParametersNAME hexadecyl deprotected carbonEXPNO 10PROCNO 1





hexadecyl deprotected carbon1d_13C{1H}_n D2O {C:\nmr_users} huestis 3

OH3C(H2C)15O

HO

OH

HO O PO

OO NH4

NH48f

S65

-10-520 15 10 5 0 ppm

-0.57

Current Data ParametersNAME Hexadecyl deprotected 3EXPNO 10PROCNO 1

F2 - Acquisition ParametersDate_ 20070719Time 1.33INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zggd30TD 65536SOLVENT D2ONS 1024DS 2SWH 98039.219 HzFIDRES 1.495960 HzAQ 0.3342887 secRG 7298.2DW 5.100 usecDE 7.29 usecTE 300.6 KD1 1.00000000 secd11 0.03000000 secTD0 4


======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -2.00 dBPL12 15.72 dBSFO2 500.1322696 MHz


Gaia-4,6-benzylidene1d_31P_d D2O {C:\nmr_users} huestis 4

OH3C(H2C)15O

HO

OH

HO O PO

OO NH4

NH48f

S66

MH_1261d_1H D2O {C:\nmr_users} huestis 15


F2 - Acquisition ParametersDate_ 20070519Time 22.08INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT D2ONS 64DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 71.8DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1



1.01.52.02.53.03.54.04.55.05.5 ppm

0.808

0.818

0.821

1.768

1.781

1.794

3.335

3.355

3.373

3.433

3.436

3.439

3.452

3.455

3.459

3.484

3.490

3.500

3.504

3.519

3.538

3.620

3.631

3.645

3.655

3.753

3.778

3.789

3.792

3.799

3.802

3.809

3.813

3.819

4.700

5.324

5.331

6.18

1.07

1.08

1.11

2.99

1.07

1.02

1.03

1.00

3.43.53.63.73.8 ppm

3.335

3.355

3.373

3.452

3.455

3.459

3.484

3.490

3.500

3.504

3.519

3.620

3.631

3.645

3.655

3.753

3.778

3.789

1.077

1.113

2.985

1.067

1.021

1.025

O(H3C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48g

S67







253035404550556065707580859095 ppm

18.616

18.638

28.240

60.714

69.297

71.767

71.824

72.236

80.058

81.745

93.971

94.015

O(H3C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48g

S68

-15-10-530 25 20 15 10 5 0 ppm

1.76







O(H3C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48g

S69

1.01.52.02.53.03.54.04.55.05.5 ppm

0.772

1.204

1.218

1.232

1.246

1.261

1.276

1.289

1.383

1.395

1.407

3.310

3.329

3.348

3.414

3.434

3.467

3.486

3.604

3.609

3.637

3.739

3.764

3.781

3.783

3.785

3.790

4.697

5.314

5.322

5.328

6.03

4.05

1.01

1.04

1.03

1.03

3.03

2.02

1.00


F2 - Acquisition ParametersDate_ 20070607Time 2.42INSTRUM Av500PROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT D2ONS 32DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2769001 secRG 57DW 50.000 usecDE 6.00 usecTE 300.0 KD1 1.00000000 secTD0 1



MH_1301d_1H D2O {C:\nmr_users} huestis 14

3.43.53.63.73.8 ppm

3.310

3.329

3.348

3.414

3.434

3.467

3.486

3.604

3.609

3.637

3.739

3.764

3.781

3.783

3.785

3.790

1.036

1.033

1.033

3.034

2.023

O(H3CH2C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48h

S70

2030405060708090 ppm10.16

10.23

22.44

22.45

40.98

60.71

69.28

71.73

71.78

72.28

75.68

81.66

94.04

94.09






MH_1301d_13C{1H}_n D2O {C:\nmr_users} huestis 14

O(H3CH2C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48h

S71

-40-35-30-25-20-15-10-535 30 25 20 15 10 5 0 ppm

1.62







O(H3CH2C)2HCH2CO

HO

OH

HO O PO

OO NH4

NH48h

Determination of pKa2 for α-D-glucopyranosyl phosphate and compounds 8b, 8d, 8e and 8g

pH2 4 6 8

HC

l (eq

)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Parameter Value Std. Error

pKa 1 0.6783 0.0547Lower Limit 20.5268 2.2855Middle Limit 1.0593 0.0031Upper Limit -0.0042 0.0048pKa 2 6.2798 0.0081

OHO

HO

OH

HO O PO

OO

pKa determination of α -D-glucopyranosyl phosphate

S73

pH2 4 6 8

HC

l (eq

)

00.20.40.60.8

11.21.41.61.8

22.22.42.6


pKa 1 2.1514 0.1808Lower Limit 3.4621 0.4900Middle Limit 1.4506 0.0281Upper Limit 0.0822 0.0304pKa 2 6.0911 0.0461

pKa determination of 3-O-Butyl-α -D-glucopyranosyl phosphate

OH3C(H2C)3O

HO

OH

HO O PO

OO

8b

S74

pH2 4 6 8

HC

l (eq

)

00.20.40.60.8

11.21.41.61.8

22.2



pKa determination of 3-O-Octyl-α -D-glucopyranosyl phosphate

OH3C(H2C)7O

HO

OH

HO O PO

OO

8d

S75

pH2 4 6 8 10

HC

l (eq

)

00.20.40.60.8

11.21.41.61.8

2



pKa determination of 3-O-Dodecyl-α -D-glucopyranosyl phosphate

OH3C(H2C)11O

HO

OH

HO O PO

OO

8e

S76

pH2 4 6 8

HC

l (eq

)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6



pKa determination of 3-O-(2-methylpropyl)-α -D-glucopyranosyl

phosphate

O(H3C)2HCH2CO

HO

OH

HO O PO

OO

8g

S77

Enzymatic Synthesis

General Procedure for nucleotidylyltransferase-catalyzed synthesis of sugar nucleotides (9a-h, 10a-h)

Enzymatic reactions were performed by the method of Timmons (MgCl2, 1.0 mM NTP, 2.0 mM S1P, 0.5EU

inorganic pyrophosphatase, 2EU nucleotidylyltransferase in Tris-HCl buffer – total 50 µL)6 except that MgCl2

was used at a final concentration of 5.5 mM and incubations were performed at 41ºC.

Figure S1. a) Selected data for nucleotidylyltransferase-catalyzed conversion of sugar-1-phosphates (8a-h) to sugar nucleotides (9a-h, 10a-h) using a) Cps2L b) RmlA3.

6 Timmons, S. C.; Mosher, R. H.; Knowles, S. A.; Jakeman, D. L. Org. Lett. 2007, 9, 857-860.

S78

Enzyme Sugar-1-Phosphate NTP

% Conv. (4 hr)

% Conv. (12 hr)

% Conv. (24 hr)

% Conv (48 hr)

Sugar Nucleotide retention time (min)

Cps2L 8a dTTP 90 100 5.82Cps2L 8b dTTP 43 89 100 6.73Cps2L 8c dTTP c c c c 7.50Cps2L 8d dTTP 19 52 85 100 8.28Cps2L 8e dTTP 36 72 95 100 9.77Cps2L 8f dTTP 32 29 19 21 9.25a

Cps2L 8g dTTP 17 42 71 100 6.75Cps2L 8h dTTP 23 54 80 100 7.35Cps2L 8a UTP b b b b 5.75Cps2L 8b UTP 3 2 3 5 6.69Cps2L 8c UTP c c 6 10 7.48Cps2L 8d UTP 2 2 3 5 8.26Cps2L 8e UTP 3 4 8 16 9.77Cps2L 8f UTP 6 7 5 7 9.27a

Cps2L 8g UTP 0 0 1 2 6.66Cps2L 8h UTP 1 c c c 7.32RmlA3 8a dTTP 28 b b b 5.82RmlA3 8b dTTP 42 100 6.73RmlA3 8c dTTP c c c c 7.50RmlA3 8d dTTP 16 41 77 100 8.28RmlA3 8e dTTP 22 64 92 100 9.77RmlA3 8f dTTP 8 7 7 21 9.25a

RmlA3 8g dTTP 14 34 56 100 6.75RmlA3 8h dTTP 22 57 93 100 7.35RmlA3 8a UTP b b 7 b 5.75RmlA3 8b UTP 11 18 30 breakdown 6.69RmlA3 8c UTP c 9 13 14 7.48RmlA3 8d UTP 5 7 12 20 8.26RmlA3 8e UTP 7 13 10 11 9.77RmlA3 8f UTP 5 7 3 9 9.27a

RmlA3 8g UTP 4 7 11 5 6.66RmlA3 8h UTP 5 c c c 7.32

Table S1. Nucleotidylyltransferase-catalyzed conversion of sugar-1-phosphates (8a-h) to sugar nucleotides (9a-h, 10a-h). aalternate buffer gradient used (see General Methods). binaccurate integration due to co-elution with NDP. cinaccurate integration due to co-elution with NTP.

S79

3-O-Methyl-dTDP-α-D-glucose (9a)

LRMS m/z calcd for C17H26N2O16P22- [M+H]-: 577.1. Found 577.0. EPI fragments: 321.1.

S80

3-O-Butyl-dTDP-α-D-glucose (9b)


S81

3-O-Hexyl-dTDP-α-D-glucose (9c)


S82

3-O-Octyl-dTDP-α-D-glucose (9d)


S83

3-O-Dodecyl-dTDP-α-D-glucose (9e)


S84

3-O-Hexadecyl-dTDP-α-D-glucose (9f)


S85

3-O-(2-Methylpropyl)-dTDP-α-D-glucose (9g)


S86

3-O-(2-Ethylbutyl)-dTDP-α-D-glucose (9h)


S87

3-O-Methyl-UDP-α-D-glucose (10a)


S88

3-O-Butyl-UDP-α-D-glucose (10b)


S89

3-O-Hexyl-UDP-α-D-glucose (10c)


S90

3-O-Octyl-UDP-α-D-glucose (10d)


S91

3-O-Dodecyl-UDP-α-D-glucose (10e)


S92

3-O-Hexadecyl-UDP-α-D-glucose (10f)


S93

3-O-(2-Methylpropyl)-UDP-α-D-glucose (10g)


S94

3-O-(2-Ethylbutyl)-UDP-α-D-glucose (10h)


obc supporting information 3-o-alkyl-glucose mh information lipophilic sugar nucleotide synthesis by...

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