Treating water stress in plants

Abstract

The present invention relates to methods and uses of photo-labile compounds which are trehalose-6-phosphate or trehalose-6-phosphonate or agriculturally acceptable salts thereof in the treatment of water stress in plants. The invention also concerns methods and the use of the compounds for resurrection of water stressed plants, and for improvement of yield of crop plants under water stressed conditions compared with untreated plants.

Claims

1. A method of increasing the yield of a drought stressed crop plant, the method comprising applying a compound to the plant, wherein the compound is of formula (I) or agriculturally acceptable salt thereof: ##STR00009## wherein; p is 0 or 1; R.sub.1 to R.sub.7 independently represent F, N.sub.3, NR′R″, C.sub.1-4alkyl, —(C.sub.1-4alkyl)OH or OH, wherein R′ and R″ independently represent hydrogen or C.sub.1-4alkyl; and R.sub.8 and R.sub.9 are the same or different and represent H or a photo-labile protecting group, wherein at least one of R.sub.8 and R.sub.9 represents a photo-labile protecting group, and wherein the photo-labile protecting group is of formula (II): ##STR00010## wherein; ring A is selected from the group consisting of a C.sub.6-10 aryl group, and a dibenzofuranyl ring, and further wherein the aryl group is unsubstituted or substituted with one or more substituents selected from C.sub.1-4 alkyl, —OR′, halogen, CN, —NR′R″, —COOR′, —(C.sub.1-4 alkyl)COOR′ and —O(C.sub.1-4 alkyl)COOR′, wherein R′ and R″ are independently selected from hydrogen and C.sub.1-4 alkyl or wherein two adjacent ring positions of the C.sub.6-10 aryl group are substituted with a —CH.sub.2—O—CH.sub.2 moiety; either (i) R.sub.10 and R.sub.11 are the same or different and are selected from hydrogen, C.sub.1-4alkyl which is unsubstituted or substituted with one or more halogen atoms, —OR′, halogen, —NR′R″ or —CO.sub.2R′, wherein R′ and R″ are independently selected from hydrogen and C.sub.1-4alkyl, or (ii) two R.sub.10 groups on adjacent photo-labile protecting groups together form a bond and R.sub.11 represents hydrogen, C.sub.1-4alkyl which is unsubstituted or substituted with one or more halogen atoms, —OR′, halogen, —NR′R″ or —CO.sub.2R′, wherein R′ and R″ are independently selected from hydrogen and C.sub.1-4alkyl; n is 0 or 1; and R.sub.12 and R.sub.13 are the same or different and are selected from hydrogen, C.sub.1-4alkyl which is unsubstituted or substituted with one or more halogen atoms, —OR′, halogen, —NR′R″ or —CO.sub.2R′, wherein R′ and R″ are independently selected from hydrogen and C.sub.1-4 alkyl; wherein X represents the link to the remainder of the compound of formula (I).

2. The method of claim 1, wherein the photo-labile group is selected from; ##STR00011## wherein X represents the link to the remainder of the compound of formula (I).

3. The method of claim 1, wherein the photo-labile protecting group is of formula (II) and ring A represents a C.sub.6-10 aryl group or a dibenzofuranyl ring, wherein the aryl is unsubstituted or substituted with one or more substituents selected from C.sub.1-4alkyl, —OR′, halogen, CN, —NR′R″, —COOR′, —(C.sub.1-4alkyl)COOR′, and —O(C.sub.1-4alkyl) COOR′, wherein R′ and R″ are independently selected from hydrogen and C.sub.1-4alkyl.

4. The method of claim 1, wherein the photo-labile protecting group is of formula (II) and ring A represents a phenyl, or naphthalenyl.

5. The method of claim 1, wherein the photo-labile protecting group is of formula (IIa): ##STR00012## wherein; ring A represents an unsubstituted or substituted group selected from phenyl, naphthyl or dibenzofuranyl, wherein a substituted phenyl, naphthyl or dibenzofuranyl group is substituted with one or two methoxy substituents or wherein two adjacent ring positions are substituted with a —CH.sub.2—O—CH.sub.2— moiety; and the R.sub.10 represents hydrogen, methyl, —CF3 or —COOH; wherein X represents the link to the remainder of the compound of formula (I).

6. The method of claim 1, wherein R.sub.1 to R.sub.7 represent hydroxyl.

7. The method of claim, wherein p is 1.

8. The method of claim 1, wherein the crop plant is a cereal crop selected from a genera selected from the group consisting of Triticum, Zea, Oryza, Hordeum, Sorghum, Panicum, Avena, and Secale.

9. The method of claim 1, wherein the compound of formula (I) is applied to the drought stressed plant at least 24 hours prior to re-watering the drought stressed plant.

10. The method of claim 1, wherein the drought stressed plant has been under drought stress prior to treatment for a period falling in the range of 8 hours to 10 days.

11. The method of claim 1, wherein the compound of formula (I) is applied to the drought stressed plant together with at least one fertilizer, fungicide, herbicide, insecticide or plant growth regulator.

12. The method of claim 1, wherein the drought stressed plant following treatment has increased growth, biomass and yield compared with corresponding untreated drought stressed plants.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will now be described in detail with reference to Examples and with reference to the accompanying drawings, in which:

(2) FIG. 1 shows a schematic diagram of a chemical strategy to control trehalose-6-phosphate (T6P) in plants.

(3) FIG. 2 shows a schematic diagram illustrating the principle of photo-activated release of T6P in planta from plant permeable signalling precursors of T6P.

(4) FIG. 3 shows the synthesis of bis-(2-nitrobenzyl)-N,N-diisopropylphosphoramidite 9.

(5) FIG. 4 shows the synthesis of bis-(4,5-dimethoxy-2-nitrobenzyl)-N,N diisopropylphosphoramidite 10.

(6) FIG. 5 shows the synthesis of bis-[1-(2-nitrophenyl)-ethyl]-N,N-diisopropylphosphoramidite 11.

(7) FIG. 6 shows the synthesis of compound 1.

(8) FIG. 7 shows the synthesis of compound 2.

(9) FIG. 8 shows the synthesis of compound 3.

(10) FIG. 9 shows the synthesis of compound 4.

(11) FIG. 10 shows the synthesis of compound 13.

(12) FIG. 11 shows the synthesis of compound 14.

(13) FIG. 12 shows the synthesis of compound 15.

(14) FIG. 13 shows the synthesis of compound 16.

(15) FIG. 14 shows the synthesis of compound 17.

(16) FIG. 15 shows the .sup.1H and .sup.13C NMR Spectra of compound 9.

(17) FIG. 16 shows the .sup.1H and .sup.13C NMR Spectra of compound 9.

(18) FIG. 17 shows the .sup.1H and .sup.13C NMR Spectra of compound 10.

(19) FIG. 18 shows the .sup.1H and .sup.13C NMR Spectra of compound 10.

(20) FIG. 19 shows the .sup.1H and .sup.13C NMR Spectra of compound 11.

(21) FIG. 20 shows the .sup.1H and .sup.13C NMR Spectra of compound 11.

(22) FIG. 21 shows the .sup.1H and .sup.13C NMR Spectra of compound 1.

(23) FIG. 22 shows the .sup.1H and .sup.13C NMR Spectra of compound 1.

(24) FIG. 23 shows the .sup.1H and .sup.13C NMR Spectra of compound 2.

(25) FIG. 24 shows the .sup.1H and .sup.13C NMR Spectra of compound 2.

(26) FIG. 25 shows the .sup.1H and .sup.13C NMR Spectra of compound 3.

(27) FIG. 26 shows the .sup.1H and .sup.13C NMR Spectra of compound 3.

(28) FIG. 27 shows the .sup.1H and .sup.13C NMR Spectra of compound 4.

(29) FIG. 28 shows the .sup.1H and .sup.13C NMR Spectra of compound 4.

(30) FIG. 29 shows the .sup.1H and .sup.13C NMR Spectra of compound 14.

(31) FIG. 30 shows the .sup.1H and .sup.13C NMR Spectra of compound 14.

(32) FIG. 31 shows the .sup.1H and .sup.13C NMR Spectra of compound 15.

(33) FIG. 32 shows the .sup.1H and .sup.13C NMR Spectra of compound 15.

(34) FIG. 33 shows the .sup.1H and .sup.13C NMR Spectra of compound 16.

(35) FIG. 34 shows the .sup.1H and .sup.13C NMR Spectra of compound 16.

(36) FIG. 35 shows the .sup.1H and .sup.13C NMR Spectra of compound 17.

(37) FIG. 36 shows the .sup.1H and .sup.13C NMR Spectra of compound 17.

(38) FIG. 37 shows the .sup.1H and .sup.13C NMR Spectra of compound T6P.

(39) FIG. 38 shows the .sup.1H and .sup.13C NMR Spectra of compound T6P.

(40) FIG. 39 shows the known biochemical structures and one-pot synthesis of designed permeable, signalling precursor variants from suitable precursors.

(41) FIG. 40 shows the effect of spraying T6P signalling precursors on crop resilience; overall phenotypes of plants after one application of 1 mM oNPE-T6P (3) or 1 mM DMNB-T6P (2) one day prior to re-watering after 20 days recovery.

(42) FIG. 41 shows the biomass from the experimental plants of FIG. 4. Asterisks indicate statistical significance.

(43) FIG. 42 shows overall phenotypes of plants after one application of 1 mM oNPE-T6P (3) or 1 mM DMNB-T6P (2) one day prior to re-watering, cut at 5 days after re-watering, and left to regrow for 10 days. Cut back point is indicated by white arrow and line. Regrowth is indicated by black arrow.

(44) FIG. 43 shows the fresh weight biomass of regrowth. Asterisks indicate statistical significance.

DETAILED DESCRIPTION

Examples

(45) The trehalose 6-phosphate (T6P) synthesis pathway in plants is summarized in FIG. 1. Photosynthesis generates sucrose, which is translocated to growing regions of the plant. Inside the cell it feeds a pool of core metabolites which are substrates for biosynthetic processes that determine growth and productivity. T6P is synthesised from UDPG and G6P by trehalose 6-phosphate synthase (TPS) and therefore reflects the abundance of sucrose. It is broken down by trehalose phosphate phosphatase (TPP). Increasing T6P (a) stimulates starch synthesis through posttranslational redox activation of ADP-glucose pyrophosphorylase (AGPase) which catalyzes the first committed step of starch biosynthesis and (b) inhibits SnRK1, a protein kinase central to energy conservation and survival during energy deprivation. Inhibition of SnRK1 by T6P thus diverts carbon skeleton consumption into biosynthetic processes.

Example 1. Design and Synthesis of Signalling Precursors of T6P

(46) T6P is plant impermeable (FIG. 2). In order to alter T6P levels in planta, plant permeable signalling precursor variants were designed and synthesised in a single pot reaction starting from suitable precursors.

(47) Synthesis of signalling-precursor compounds 1-4

(48) 1H-tetrazole solution (0.45 M in CH.sub.3CN) (0.6 mL, 0.24 mmol, 2.0 equiv.) was added into a stirred solution of 12 (100 mg, 0.12 mmol, 1 equiv.) and bis-(2-nitrobenzyl)-N,N-diisopropylphosphoramidite 9 (78.3 mg, 0.18 mmol, 1.5 equiv.) in anhydrous CH.sub.2Cl.sub.2 (5 mL) under an argon atmosphere at 0° C. The resulting reaction mixture was stirred at 0-5° C. and progress of the reaction was monitored by TLC (petroleum ether:ether; 8:2) and mass spectrometry. After complete disappearance of starting material (1 h), tBuOOH (0.1 mL) was added at 0° C. and stirring was continued for another 30 min. After 30 min the reaction mixture was concentrated in vacuo and the residue was suspended in methanol (2 mL) and stirred in the presence of 30 mg of Dowex-H.sup.+ resin for 1 h at room temperature to globally remove TMS groups.

(49) Dowex-H.sup.+ was removed through filtration and the filtrate was concentrated, which on flash chromatography (water:isopropanol:ethyl acetate, 1:2:8) purification yielded 1 (70 mg) in 87% isolable yield. Similar reaction protocols were adopted for the synthesis of compounds 2 and 3. Compound 4 was obtained when a stirred solution of 12 (100 mg, 0.12 mmol) in pyridine (2 mL) at room temperature was treated with POCl.sub.3 (0.012 mL, 0.132 mmol) for 10 min followed by addition of 4,5-dimethoxy-2-nitrobenzyl alcohol (76.7 mg, 0.36 mmol) and continuous stirring for 1 h.

(50) The resulting reaction mixture was concentrated in vacuo to yield crude product mixture, which was treated with Dowex-H.sup.+ (30 mg) in methanol (2 mL). After filtration, concentration in vacuo and flash chromatography purification yielded 4 (45 mg, 62%) as a pure sticky solid. Full details of the synthesis of each of the compounds are provided below.

Synthetic Protocols, Experimental and Characterization Data for all Compounds

Synthesis of Bis-2-Nitrobenzyl-N,N-Diisopropylphosphoramidite 9

(51) Diisopropylphosphoramidous dichloride 5 (2.0 g, 9.90 mmol) was dissolved in 15 mL of THF and the resulting solution was added slowly to a solution containing 4.2 mL (29.7 mmol) of triethylamine and 3.03 g (19.8 mmol) of 2-nitrobenzyl alcohol 6 in 10 mL of THF at 0° C. The reaction mixture was stirred at 0° C. for 30 min and then at 25° C. for another 2 h. The colorless precipitate was isolated by filtration and the solid was washed with 100 mL of ethyl acetate. The organic phase was washed successively with 15 mL portions of saturated NaHCO.sub.3 and saturated NaCl and then dried (MgSO4) and concentrated under reduced pressure at 25° C. The residue was precipitated from ethyl acetate/hexane, affording bis (2-nitrobenzyl) N,N-diisopropylphosphoramidite 9 (3.0 g, 70%) as a colorless solid..sup.5 (see FIG. 3).

bis-(2-nitrobenzyl)-N,N-diisopropylphosphoramidite 9

(52) Mp 71-72° C. [lit..sup.5 Mp 71-73° C.]1H NMR (400 MHz, CDCl.sub.3) δ 8.10 (d, J=8.4 Hz, 2H, H-3 and H-3′), 7.86 (d, J=8.0 Hz, 2H-6 and H-6′), 7.67 (t, J=8.0 Hz, 2H, H-5 and H-5′), 7.44 (t, J=8.4 Hz, 2H, H-4 and H-4′), 5.21 (dd, J=16.4 Hz and J=6.8 Hz, 2H, CH.sub.2Ar), 5.12 (dd, J=16.4 Hz and J=6.8 Hz, 2H, CH.sub.2Ar), 3.77-3.71 (m, 2H, 2×CH(CH.sub.3).sub.2), 1.25 (d, J=8.5 Hz, 12H, 2×CH(CH.sub.3).sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3) 146.8 (C-2 and C-2′), 136.1, 136.0 (C-I and C-I′), 133.7 (C-6 and C-6′), 128.5 (C-5 and C-5′), 127.8 (C-4 and C-4′), 124.6 (C-3 and C-3′), 62.5 (CH.sub.2Ar), 62.3 (CH.sub.2Ar), 43.4 (CH(CH.sub.3).sub.2), 43.3 (CH(CH.sub.3).sub.2), 24.7 (CH(CH.sub.3).sub.2), 24.6 (CH(CH.sub.3).sub.2); .sup.31P NMR (162 MH, CDCl.sub.3) δ 149.0; ESI-LRMS m/z calculated for C.sub.20H.sub.26N.sub.3O.sub.6P [M+H].sup.+ 436.1; Found 436.1.

Synthesis of bis-(4,5-dimethoxy-2-nitrobenzyl)-N,N-diisopropylphosphoramidite 10

(53) To a −20° C. cooled suspension of 4,5-dimethoxy-2-nitrobenzyl alcohol 7 (2.1 g, 9.90 mmol) and triethylamine (1.5 mL, 10.8 mmol) in dry THF (10 mL) was added dropwise a solution of diisopropylphosphoramidous dichloride 5 (1.0 g, 4.95 mmol) in dry THF (2 mL). The mixture was allowed to warm to 20° C., stirred for 18 h, and a saturated solution of aq. NaHCO.sub.3, (15 mL) added. The solid was filtered, washed with water (20 mL) and dried to give 2.0 g (74%) of 10.sup.6. (see FIG. 4)

bis-(4,5-dimethoxy-2-nitrobenzyl)N,N-diisopropylphosphoramidite 10

(54) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.72 (s, 2H, H-3 and H-3′), 7.39 (s, 2H, H-6 and H-6′), 5.24 (dd, J=16.4 Hz and J=6.8 Hz, 2H, CH.sub.2Ar), 5.15 (dd, J=16.4 Hz and J=6.8 Hz, 2H, CH.sub.2Ar), 3.95 (s, 6H, 2×OMe), 3.94 (s, 6H, 2×OMe), 3.85-3.70 (m, 2H, 2×CH(CH.sub.3).sub.2) 1.27 (d, J=8.5 Hz, 12H 2×CH(CH.sub.3).sub.2); .sup.13C NMR (100 MHz, CDCl.sub.3) 153.8 (C-5 and C-5′), 147.5 (C-4 and C-4′), 138.6 (C-2 and C-2′), 131.7, 131.6 (C-3 and C-3′), 109.2 (C-1 and C-1′). 107.8 (C-6 and C6′), 62.5 (CH.sub.2Ar), 62.4 (CH.sub.2Ar), 56.3 (OMe). 43.4 (CH(CH.sub.3).sub.2), 43.3 (CH(CH.sub.3).sub.2), 25.6 (CH(CH.sub.3).sub.2), 24.7 (CH(CH.sub.3).sub.2); .sup.31NMR (162 MHz, CDCl.sub.3) 147.4; ESI-LRMS m/z calculated for C.sub.24H.sub.34N.sub.3O.sub.10P [M+H].sup.+: 556. L; Found 556.1.

Synthesis of bis-[1-(2-nitrophenyl)-ethyl]N,N-diisopropylphosphoramidite 11

(55) Diisopropylphosphoramidous dichloride 5 (1.0 g, 4.95 mmol) was dissolved in 5 mL of dry THF and the resulting solution was added slowly to a solution containing 1.5 mL (10.89 mmol) of triethylamine and 1.65 g (9.90 mmol) of 1-methyl-2-nitrobenzyl alcohol 8 in 10 mL of THF at 0° C., The reaction mixture was stirred at 0° C. for 1 min and then at 25° C. for another 18 h. The reaction mixture was diluted with EtOAc. The organic phase was washed successively with 15 mL portions of saturated NaHCO.sub.3 and saturated NaCl and then dried (MgSO4) and concentrated under reduced pressure at 25° C. to get crude product. The residue was purified by flash column chromatography using ethyl acetate/petroleum ether (5:95 v/v), affording bis-[1-(2-nitrophenyl)-ethyl]-N, N-diisopropylphosphoramidite 11 (1.6 g, 72%) as a colorless solid. (see FIG. 5)

Bis-[1-(2-nitrophenyl)-ethyl)-N, N-diisopropylphosphoramidite 11

(56) Isolated as a dia-stereomeric mixture. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.83-7.76 (m, 3H, H-3, H-3′ and H-5), 7.54-7.46 (m, 3H, H-5′, H-4 and H-4′), 7.33-7.18 (m, 2H, H-6 and H-6′). 5.48-5.29 (m, 2H, CH(CH.sub.3)Ar), 3.62-3.44 (m, 2H, 2×CH(CH.sub.3).sub.2), 1.55-1.48 (m, 3H, CH(CH.sub.3)Ar), 1.40-1.35 (m, 3H, CH(CH.sub.3)Ar), 1.13-1.07 (m, 6H, CH (CH.sub.3).sub.2), 0.90-0.83 (m, 6H, CH(CH.sub.3).sub.2).sub.; .sup.13C NMR (100 MHz, CDCl.sub.3).sup.5 147.2, 147.1, 146.9 (C-2 and C-2′), 141.3. 141.1, 140.8 (C-1 and C-1′), 133.4, 133.3 (C-3 and C-3′), 128.5, 128.3 (C-5 and C-5′), 127.8, 127.7 (C-4 and C-4′), 124.0, 123.9 (C-6 and C-6′), 67.3, 67.2 (CH(CH.sub.3)Ar), 67.0, 66.7 (CH(CH.sub.3), 43.1. 43.0, 25.1, 25.0 (CH(CH.sub.3).sub.2), 24.5, 24.4 (CH(CH.sub.3).sub.2), 24.2. 24.1 (CH(CH.sub.3)Ar); ESI-LRMS m/z calculated for C.sub.22H.sub.30N.sub.3O.sub.6P [M+11].sup.+; 464.1; Found 464.1.

Synthesis of Compound 1

(57) To a solution of 12 (100 mg, 0.12 mmol, 1 equiv.) and 1H-tetrazol solution (16.8 mg, 0.24 mmol, 2.0 equiv. ˜0.5 mL of 0.4 M solution in CH.sub.3CN) in anhydrous CH.sub.2Cl.sub.2 (4 mL) under an argon atmosphere at 0° Cm bis-(2-nitrobenzyl) N,N-diisopropylphosphoramidite 9 (78.3 mg, 0.18 mmol, 1.5 equiv.) was added. The solution was stirred for 30 min and progress of the reaction was monitored by TLC (petroleum ether:ether; 8:2). After complete disappearance of starting material, tBuOOH (32.5 mg, 0.36 mmol, 3.0 equiv ˜0.1 mL of 5 M solution in decane) was added at 0° C. and stirred for 30 min. After 30 min the reaction mixture was concentrated in vacuo and the residue was stirred with 30 mg of Dowex-H.sup.+ resin in methanol (10 mL) for 1 h to obtain deprotected compound as a crude product. This crude product after flash chromatography purification yielded desired product 1 (70 mg) in 87% isolable yield. (see FIG. 6)

6-O-bis-(2-nitrobenzyloxyphosphoryl)-D-trehalose 1

(58) R.sub.f0.60 (1 water:2 isopropanol:4 ethyl acetate); [α].sub.D.sup.21+80.6 (c 1.0, MeOH); FT-IR (ATR) ν cm.sup.−1 (3347 (br, OH), 1526 (s, N═O), 1343 (s, N═O), 1255 (P═O); .sup.1H NMR (500 MHz, D.sub.2O) δ 8.02 (d, J=8.0 Hz, 2H ArH), 7.66-7.65 (m, 4H, ArH), 7.50-7.46 (m, 2H, ArH), 5.43 (d, J=7.2 Hz, 4H, 2×(CH.sub.2Ar), 4.96 (d, J.sub.1′,2′=3.6 Hz, 1H, H-1′), 4.93 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 4.40 (dd, J.sub.6′a,6′b=11.0 Hz, J.sub.6′a,5=2.0 Hz, 1H, H-6′a), 4.35 (dd, J.sub.6′B,6′a=11.0 Hz, J.sub.6′b,5=4.5 Hz, 1H, H-6′b), 3.93 (td, J.sub.5′4′=10.0 Hz and J.sub.5′,6′a=2.0 Hz, 1H, H-5′), 3.71 (t, J.sub.32′=9.2 Hz, J.sub.3′,4′=9.2 Hz, 1H, H-3′), 3.70-3.68 (m, 1H, H-5), 3.67 (t, J.sub.3,2=9.6 Hz, J.sub.3,4=9.6 Hz, 1H, H-3), 3.66-3.65 (m, 1H, H-6a), 3.58 (dd, J.sub.6b,6a=12.0 Hz and J.sub.6b,5=5.2 Hz, 1H, H-6b), 3.44 (dd, J.sub.2′3′=9.9 Hz, J.sub.2′,1′=3.5 Hz, 1H, H-2′), 3.40 (dd, J.sub.2,3=9.6 Hz, J.sub.2,1=3.8 Hz, 1H, H-2), 3.27 (t, J.sub.4′3′=9.6 Hz, J.sub.4′5′=9.6 Hz, 1H, H-4′), 3.22 (t, J.sub.4,3=9.6 Hz, J.sub.4,5=9.6 Hz, 1H, H-4); .sup.13C NMR (125 MHz, D20) δ 147.7 (qCAr), 134.3 (qCAr), 132.1 (ArC), 132.0 (ArC), 129.4 (ArC), 129.0 (ArC), 128.9 (ArC), 125.0 (ArC), 94.4 (C-1′), 94.3 (C-1), 73.5 (C-3′), 73.3 (C-3), 72.8 (C-2′), 72.1 (C-2), 72.0 (C-5′), 71.0 (C-5), 70.9 (C-4′), 70.8 (C-4), 70.1 (C-6′), 67.2 (CH.sub.2Ar), 66.6 (CH.sub.2Ar), 61.6 (C-6); .sup.31P NMR (162 MHz, D.sub.2O) δ −0.11; ESI-HRMS m/z calculated for C.sub.26H.sub.33N.sub.2O.sub.18P [M+Na].sup.+715.1368; Found 715.1368.

Synthesis of Compound 2

(59) To a solution of 12 (100 mg, 0.12 mmol, 1 equiv.) and 1H-tetrazol (84 mg, 1.2 mmol, 10 equiv. 3.0 mL of 0.4 M solution in CH.sub.3CN) in anhydrous CH.sub.2Cl.sub.2 (8 mL) under an argon atmosphere at 0° C., bis-(4,5-dimethoxy-2-nitrobenzyl)-N,N-diisopropylphosphoramidite 10 (100 mg, 0.18 mmol, 1.5 equiv.) was added and the resulting reaction mixture was stirred at 0-5° C. The progress of the reaction was monitored by TLC (petroleum ether:ether; 8:2). After complete disappearance of starting material (18 h), tBuOOH (32.5 mg, 0.36 mmol, 3.0 equiv. ˜0.1 mL of 5 M solution in decane) was added at 0° C. and the mixture stirred for a further 30 min. After 30 min the reaction mixture was concentrated in vacuo and the residue was stirred with 30 mg of Dowex-H.sup.+ resin in methanol (10 mL) for 1 h to obtain deprotected compound as crude product. This crude product after flash chromatography purification yielded desired product 2 (50 mg) in 52% isolable yield. (see FIG. 7)

6-O-bis-(4,5-dimethoxy-2-nitrobenzyloxyphosphoryl)-D-trehalose 2

(60) R.sub.f0.50 (1 water; 2 isopropanol:4 ethyl acetate); [α].sub.D.sup.21+64.8 (c 1.1, MeOH); FT-IR (ATR) ν cm.sup.−1 3347 (br, OH), 1519 (s, N═O), 1326 (s, N═O), 1220 (P═O): .sup.1H NMR (500 MHz, CD.sub.3OD) β 7.53 (s, 2H, ArH), 7.03 (s, 2H, ArH), 5.37 (d, J=8.0 Hz, 4H, 2×CH2Ar), 4.95 (d, J.sub.1′2′=4.0 Hz, 1H, H-1′), 4.91 (d, J.sub.1,2=4.0 Hz, 1H, H-1), 4.30 (dd, J.sub.6′a, 6′b=11.6 Hz, J.sub.6′a,5=2.0 Hz, IH, H-6′a), 4.35 (dd, J.sub.6′b,6′a=11.0 Hz, J.sub.6b,5=3.6 Hz, 1H. H-6′b), 3.94 (td, =10.0 Hz and J.sub.5′,6′a=2.0 Hz, 1H, H-5′), 3.71 (t, J.sub.3′,2′=9.6 Hz, J.sub.3′,4′=9.6 Hz, 1H, H-3′), 3.70-3.68 (m, 1H, H-5), 3.67 (t, J.sub.32=9.6 Hz, J.sub.3,4=9.6 Hz, 1H, H-3), 3.66-3.65 (m, 1H, H-6a), 3.58 (dd, J.sub.6b,6a=11.6 Hz and J.sub.6b,5=5.6 Hz, 1H, H-6b), 3.35 (dd, J.sub.2′3′=8.4 Hz, J.sub.2′,1′=4.0 Hz, 1H, H-2′), 3.33 (dd, J.sub.2,3=8.5 Hz, J.sub.2.1=3.8 Hz, 1H, H-2), 3.26 [t, J.sub.4′,3′=8.8 Hz, J.sub.4′5′=8.8 Hz, 1H, H-4′), 3.24 (t, J.sub.4,3.sup.=9.6 Hz, J.sub.4.5=9.6 Hz, 1H, H-4); .sup.13C NMR (125 MHz, CD.sub.3OD) 154.2 (qCAr), 148.9. (qCAr), 143.7 (qCAr), 139.6 (ArC), 126.8 (ArqC), 126.6 (ArC), 110.4 (ArC), 110.3 (ArC), 108.2, 94.4 (C-1′), 94.3 (C-1), 73.5 (C-3′), 73.3 (C-3), 72.8 (C-2′), 72.1 (C-2), 72.0 (C-5′), 70.8 (C-5), 70.2 (C-6′), 69.7 (CH.sub.2Ar), 66.6 (CH.sub.2Ar), 61.6 (C-6), 56.1 (2×OMe), 55.8 (2×OMe); .sup.31P NMR (162 MHz CD.sub.3OD) δ −0.15; ESI-HRMS m/z calculated for C.sub.30H.sub.41N.sub.2O.sub.22P [M+Na].sup.+; 835.1786; Found 835.1782.

Synthesis of Compound 3

(61) To a solution of 12 (100 mg, 0.12 mmol, 1 equiv.) and 1H-tetrazol (84 mg, 1.2 mmol, 10 equiv. 3.0 mL of 0.4 M solution in CH.sub.3CN) in anhydrous CH.sub.2Cl.sub.2 (8 mL) under an argon atmosphere at ° C., bis-[1-(2-nitrophenyl)-ethyl]-N,N-diisopropylphosphoramidite 11 (83.5 mg, 0.18 mmol, 1.5 equiv.) was added and the resulting reaction mixture was stirred at 0-5° C. The progress of the reaction was monitored by TLC (petroleum ether:ether; 8:2) and mass spectrometry. After complete disappearance of starting material (18 h), tBuOOH (32.5 mg, 0.36 mmol, 3.0 equiv. ˜0.1 mL of 5 M solution in decane) was added at 0° C. and stirred for 30 min. After 30 min the reaction mixture was concentrated in vacuo and the residue was stirred with 30 mg of Dowex-H.sup.+ resin in methanol (10 mL) for 1 h to obtain deprotected compound as crude product. This crude product after flash chromatography purification yielded desired product 3 (44 mg) in 52% isolable yield. (see FIG. 8)

6-O-bis[1-(2-nitrophenyl)-ethoxyphosphoryl]-D-trehalose 3

(62) Isolated as mixture of four diastereomers. R.sub.f0.65 (1 water:2 isopropanol:4 ethyl acetate); FT-IR (ATR) ν cm.sup.−1 3394 (br, OH), 1521 (s, N═O), 1326 (s, N═O), 1276 (P═O); .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.91-7.05 (m, 8H, ArH), 5.92-5.84 (m, 2H, 2×CHMe), 5.04-4.94 (in, 2H, H-1 and H-1′), 3.90-3.60 (m, 7H), 3.41-3.05 (m, 5H), 1.56-1.46 (m, 6H, 2×CHMe); .sup.13C NMR (125 MHz, CD.sub.3OD δ 148.8, 148.3, 148.2 148.1 (qCAr), 147.9, 145.4, 138.9, 138.3 (qCAr), 138.2, 135.4, 135.3, 135.2 (ArC), 130.4, 130.3, 129.9, 129.3, 129.2, 128.8 (ArC) 128.7, 128.6, 126.3, 125.6, 125.5 (ArC), 95.4 (C-1′), 95.3 (C-1), 79.8, 74.6 (C-3′), 74.4, 74.2, (C-3), 74.0, 73.9 (C-2′), 73.6, 73.3 (C-2), 73.2, 73.1 (C-5′), 73.0, 72.9 (C-5), 71.9, 71.8 (C-4′), 71.2, 71.1 (C-4), 71.0 (C-6′), 68.5, 68.4 (CHMe.sub.2), 68.2, 68.1 (CHMe.sub.2), 62.6, 62.1 (C-6), 30.7, 30.5, 24.7 (CHCH.sub.3), 24.6, 23.5, 23.7 (CHCH.sub.3); .sup.31P NMR (162 MHz, CD.sub.3OD) δ −1.70, −2.20, −2.50, −2.81 (P═O) four peaks from different diastereometers; ESI-HRMS m/z calculated for C.sub.28H.sub.37N.sub.2O.sub.18P [N+Na].sup.+: 743.1677; Found 743.1676.

Synthesis of Compound 4

(63) To a stirred solution of compound 12 (100 mg, 0.12 mmol) in pyridine (2 mL) at room temperature, POCl.sub.3 (0.012 mL, 0.132 mmol) was added dropwise and the mixture was stirred for a further 10 min. After 10 min 4,5-dimethoxy-2-nitrobenzyl alcohol (DMNB-OH) (76.7 mg, 0.36 mmol) was added and the reaction mixture stirred for further 1 h. The reaction mixture was concentrated in vacuo to get crude product mixture, which after treatment with Dowex-H.sup.+ (50 mg) in methanol (2 mL) furnished compound 2 and 4. After filtration, concentration in vacuo and flash chromatography purification yielded 4 (45 mg, 62%) as a gum. (see FIG. 9)

6-O-(4,5-dimethoxy-2-nitrobenzyloxyphosphoryl)-D-trehalose 4

(64) R.sub.f 0.33 (1 water:2 isopropanol:4 ethyl acetate); [α].sub.D.sup.21+48.7 (c 1.1, MeOH); FT-IR (ATR) ν cm.sup.−1 3312 (br, OH), 1521 (s, N═O), 1326 (s, N═O), 1220 (P═O); .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.62 (s, 1H, ArH), 7.39 (s, 1H, ArH), 5.21 (d, J=6.0 Hz, 2H, CH.sub.2Ar), 4.91 (d, J.sub.1,2=4.0 Hz, 1H, H-1′), 4.87 (d, J.sub.1,2=4.0 Hz, 1H, H-1), 4.14-3.98 (m, 2H, H-6′), 3.88 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.71-3.65 (m, 4H, H-6a, H-3′, H-3 and H-5′), 3.57 (dd, J.sub.6b,6a=12.0 Hz and J.sub.6b,5=5.6 Hz, 1H, H-6b), 3.35 (dd, =7.2 Hz and J.sub.21=3.6 Hz, 1H, H-2′), 3.32 (dd, J.sub.2,3=6.8 Hz and J.sub.2,1=3.4 Hz, 1H, H-2), 3.22-3.21 (m, 3H, H-4′, H-4 and H-5); .sup.13C NMR (125 MHz, CD.sub.3OD) δ 155.5 (qC Ar), 149.0 (qC Ar) 139.9 (qC Ar), 132.1 (qC Ar), 110.8 (ArC), 109.0 (ArC), 95.3 (C-1′), 95.2 (C-1), 74.4 (C-3′), 74.3 (C-3), 73.7 (C-2′), 73.2 (C-2), 73.1 (C-5′), 72.7 (C-5), 71.8 (C-4′), 71.4 (C-4), 65.9 (CH.sub.2Ar), 65.5 (C-6′), 62.6 (C-6), 57.0 (OMe), 56.8 (OMe); .sup.31P NMR (162 MHz, CD.sub.3OD) δ 2.18 (P═O): ESI-HRMS m/z calculated for C.sub.21H.sub.32NO.sub.18P [M−H].sup.−: 616.1279; Found 616.1273.

Synthesis of Compound 13

(65) Methyl tetra-O-trimethylsilyl-α-D-glucopyranoside (3.0 gm, 4.14 mmol, 1 equiv.) was dissolved in methanol (50 mL) and kept at 0° C. followed by the addition of K.sub.2CO.sub.3 solution in MeOH (50 mL, 4.5 g/L) at 0-4° C. and stirred for 1 h (TLC, EtOAc:petroleum ether; 1:4). After neutralization with AcOH (5 mL), the mixture was concentrated to yield crude product mixture. The crude product mixture was dissolved in dichloromethane (50 mL) and washed with water (3×15 mL). The dichloromethane layer was separated and concentrated in vacuo. Flash chromatography (EtOAc:petroleum ether; 1:9) yielded desired product 13 (1.56 g, 61%)..sup.2 (see FIG. 10)

Methyl 2,3,4-tri-O-trimethylsilyl-α-D-glucopyranoside 13

(66) colourless solid [α].sub.D.sup.21+95.3 (c 1, CHCl.sub.3), [lit.sub.2 [α].sub.D.sup.21+93 (c3, CHCl.sub.3)]; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.61 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 3.78-3.74 (m, 2H, H-6′ and H-3), 3.68 (dd, J.sub.6,5=4.4 Hz J.sub.6,6=11.6 Hz, 1H, H-6), 3.57 (ddd, J.sub.5,4=9.6 Hz, J.sub.5,6=4.3 Hz, J.sub.5,6′=3.1 Hz, 1H, H-5), 3.48 (dd, J.sub.2,1=3.0 Hz, J.sub.2,3=8.4 Hz, 1H, H-2), 3.45 (dd, J=6.4 Hz, J=2.4 Hz, 1H, H-4), 3.34 (s, 3H, OMe), 0.17 (s, 9H, Si(CH.sub.3).sub.3), 0.15 (s, 9H, Si(CH.sub.3).sub.3), 0.14 (s, 9H, Si(CH.sub.3).sub.3); .sup.13C NMR (100 MHz, CDCl.sub.3); δ 99.6 (C-1), 74.8 (C-3), 73.7 (C-2), 71.9 (C-4), 71.5 (C-5), 61.8 (C6), 54.8 (OMe), 1.2 (s, 3C, Si(CH.sub.3).sub.3), 0.86 (s, 3C, Si(CH.sub.3).sub.3), 0.46 (s, 3C, Si(CH.sub.3).sub.3); ESI-LRMS m/z calculated for C.sub.16H.sub.38O.sub.6SI.sub.3 [M+Na].sup.+: 433.18; Found 433.20.

Synthesis of Compound 14

(67) To a solution of 13 (100 mg, 0.24 mmol, 1 equiv.) and 1H-tetrazol (85 mg, 1.21 mmol, 5.0 equiv. 3.0 mL of 0.4 M soln in CH.sub.3CN) in dry CH.sub.2Cl.sub.2 (8 mL) under an argon atmosphere at 0° C., bis-(2-nitrobenzyl)-N,N-diisopropylphosphoramidite 9 (156 mg, 0.36 mmol, 1.5 equiv.) was added. The solution was stirred overnight at 0-5° C. After complete disappearance of starting material (18 h), tBuOOH (64.8 mg, 0.72 mmol, 3.0 equiv. ˜0.2 ml of 5.0 M soln in decane) was added at 0° C. After 30 min of stirring the mixture was concentrated to dryness. The residue was dissolved in methanol (15 mL) and stirred with 30 mg of Dowex-H.sup.+ resin for 1 h to obtain deprotected compound. After 1 h the mixture was filtered and the filtrate was concentrated to yield deprotected crude product which on flash chromatography purification yielded desired product 14 (66 mg) in 50% isolable yield. (see FIG. 11)

Methyl 6-O-bis-(2-nitrobenzyloxyphosphoryl)-α-D-glucopyranoside 14

(68) R.sub.f0.50 (1 Methanol: 9 dichloromethane); [α].sub.D.sup.21+49.5 (c 1.0, MeOH); FT-IR (ATR) ν cm.sup.−1 3354 (br, OH), 1525 (s, N═O), 1342 (s, N═O), 1255 (P═O); .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.00 (d, J=8.0 Hz, 2H, ArH), 7.67-7.61 (m, 4H, ArH), 7.48 (t, J=8.0 Hz, 1H, ArH), 7.47 (t, J=8.0 Hz, 1H, ArH), 5.44 (d, J=7.2 Hz, 4H, 2×CH.sub.2Ar), 4.50 (d, J.sub.1,2=3.6 Hz, 1H, HA), 4.31 (ddd, J.sub.6a,6b=11.2 Hz, J.sub.6a,31P=6.4 Hz, J.sub.6a,5=1.6 Hz, 1H, H-6a), 4.22 (ddd, J.sub.6b,6a=12.0 Hz, J.sub.6b,31P=7.2 Hz, J.sub.6b,5=4.8 Hz, 1H, H-6b), 3.57 (ddd, J.sub.5,4=10.0 Hz, J.sub.5,6=4.8 Hz, J.sub.5,6′=1.6 Hz, 1H, H-5) 3.50 (brt, J.sub.3,2=9.2 Hz J.sub.3,4=9.2 Hz, 1H, H-3), 3.24 (dd, J.sub.2,1=3.6 Hz, J.sub.2,3=9.2 Hz, 1H, H-2), 3.23 (s, 3H, OMe), 3.20 (dd, J.sub.4,3=9.2 Hz, J.sub.4,5=9.7 Hz, 1H, H-4); 13C NMR (400 MHz, CD.sub.3OD): δ 147.3 (qC Ar), 143.3 (qC Ar), 134.2, 132.0, 129.4, 128.8, 125.0

(69) (ArC), 100.3 (C-1), 73.9 (C-3), 72.3 (C-2), 70.7 (C5), 70.1 (C-4), 67.9 (C-6), 66.5 (CH.sub.2Ar), 54.7 (OMe); .sup.31P NMR (162 MHz, CD.sub.3OD) δ −1.65; ESI-HRMS m/z calculated for C.sub.21H.sub.25N.sub.2O.sub.13P [M+Na].sup.+: 567.0986; Found 567.0983.

Synthesis of Compound 15

(70) To a solution of 13 (100 mg, 0.24 mmol, 1 equiv.) and 1H-tetrazol (85 mg, 1.21 mmol, 5.0 equiv, 3.0 mL of 0.4 M soln in CH.sub.3CN) in dry CH.sub.2Cl.sub.2 (8 mL) under an argon atmosphere at 0° C., bis-(4,5-dimethoxy-2-nitrobenzyl)-N,N-diisopropylphosphoramidite 10 (200 mg, 0.36 mmol, 1.5 equiv.) was added. The solution was stirred overnight at 0-4° C. After complete disappearance of starting material (18 h), tBuOOH (64.8 mg, 0.72 mmol, 3.0 equiv. ˜0.2 mL of 5.0 M soln in decane) was added at 0° C. After 30 min of stirring the mixture was concentrated to dryness. The residue was dissolved in methanol (15 mL) and stirred with 30 mg of Dowex-H.sup.+ resin for 1 h to obtain deprotected compounds. After 1 h the mixture was filtered and the filtrate was concentrated to yield fully deprotected crude product which on flash chromatography yielded desired product 15 (60 mg) in 37% isolable yield. (see FIG. 12)

Methyl 6-O-bis-(4,5-dimethoxy-2-nitrobenzyloxyphosphoryl-α-D-glucopyranoside 15

(71) R.sub.f0.40 (1 Methanol: 9 dichloromethane); [α].sub.D.sup.21+40.7 (c 1.09, MeOH); FT-IR (ATR) ν cm.sup.−1 3355 (br, OH), 1519 (s, N═O), 1326 (s, N═O), 1220 (P═O); .sup.1H NMR (400 MHz, CD.sub.3OD): δ 7.51 (s, 2H, ArH), 7.03 (s, 2H, ArH), 5.37 (d, J=8.0 Hz, 4H, 2×CH.sub.2Ar), 4.50 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 4.32 (ddd, J.sub.6a,6b=11.2 Hz, J.sub.6a,31P=6.4 Hz, J.sub.60=1.6 Hz, 1H, H-6a), 4.22 (ddd, J.sub.6b,6a=12.0 Hz, J.sub.6b,31P=7.2 Hz, J.sub.6N5=4.8 Hz, 1H, H-6b), 3.80 (s, 3H, OMe), 3.78 (s, 3H, OMe), 3.57 (dd, J.sub.5,4=10.0 Hz, J.sub.5,6b=4.8 Hz, 1H, H-5), 3.50 (brt, J.sub.3,2=9.2 Hz, J.sub.3,4=9.2 Hz, 1H, H-3), 3.25 (dd, J.sub.2,1=3.6 Hz, J.sub.2,3=9.2 Hz, 1H, H-2), 3.24 (s, 3H, OMe), 3.21-3.16 (m, 1H, H-4); .sup.13C NMR (400 MHz, CD.sub.3OD): δ 154.1, 148.9 (qC Ar), 143.2, 139.5 (qC Ar), 126.6 110.3, 108.1 (ArC), 100.3 (C-1), 73.9 (C-3), 72.3 (C-2), 70.7 (C-5), 70.1 (C-4), 68.0 (C-6), 66.8 (CH.sub.2AR), 56.0, 55.8 (OMe), 54.7 (OMe); .sup.31P NMR (162 MHz, CD.sub.3OD) δ −1.62; ESI-HRMS m/z calculated for C.sub.25H.sub.33N.sub.2O.sub.17P [M+Na].sup.+ 687.1409; Found 687,1421.

Synthesis of Compound 16

(72) To a solution of 13 (100 mg, 0.24 mmol, 1 equiv.) and 1H-tetrazol (85 mg, 1.21 mmol, 5.0 equiv, 3.0 mL of 0.4 M sole in CH.sub.3CN) in dry CH.sub.2Cl.sub.2 (5 mL) under an argon atmosphere at 0° C., bis-[1-(2-nitrophenyl)-ethyl]-N,N-diisopropylphosphoramidite 11 (167 mg, 0.36 mmol, 1.5 equiv.) was added. The solution was stirred overnight (15 h) at 0-4° C. After complete disappearance of starting material, t-BuOOH (64.8 mg, 0.72 mmol, 3.0 equiv ˜0.2 ml of 5.0 M soln in decane) was added at 0° C. After 30 min of stirring the mixture was concentrated in vacuo. The residual mixture was deprotected by stirring in methanol (15 mL) with 25 mg of Dowex-H.sup.+ resin for 1 h. After filtration the filtrate was concentrated to yield fully deprotected crude product which on flash chromatography purification yielded desired product 16 (62 mg) in 45% yield. (see FIG. 13)

Methyl 6-O-bis[1-(2-nitrophenyl)-ethoxyphosphoryl]-α-D-glucopyranoside 16

(73) R.sub.f0.55 (1 Methanol: 9 dichloromethane); Isolated as a mixture of four diastereomers, FT-IR (ATR) ν 3334 cm.sup.−2 (br, OH), 1520 (s, N═O), 1325 (s, N═O), 1219 (P═O); .sup.1H NMR (400 MHz, CD.sub.3OD); δ 7.86-7.84 (m, 2H, ArH), 7.75-7.50 (m, 3H, ArH), 7.45-7.34 (m, 3H, ArH), 5.89-5.80 (m, 2H, 2×CH(CH.sub.3), 4.57-4.45 (4d, J.sub.1,2=3.6 Hz, 1H, H-1), 4.16-3.89 (m, 2H, H-6), 3.51-3.40 (m, 2H, H-5 and 11-3), 3.31-3.25 (m, 1H, H-2), 3.25, 3.22 3.17, 3.13 (4s, 3H, OMe), 3.16-3.12 (m, 1H, H-4), 1.68-1.57 (4d, J=6.8 Hz, 6H, 2×CH(CH.sub.3): .sup.13C NMR (400 MHz, CD.sub.3OD): δ 137.2, 137.2, 134.3, 134.2, 129.4, 129.3, 127.8, 127.7, 127.6, 127.5, 124.6, 124.5 (ArC), 100.7, 100.3, 100.2, 100.1 (C-1), 74.0, 73.9 (C-3), 73.3, 73.2 (C-2), 72.9, 72.3 (C-5), 72.2, 70.6 (C-4), 70.5, 70.1 (C-6), 70.0, 67.4 (CH(CH.sub.3)), 55.1, 54.8, 54.7, 54.6 (OMe), 23.6, 23.5, 23.4 (CH(CH.sub.3)); .sup.31P NMR (162 MHz, CD.sub.3OD) δ −3.2, −3.7, −3.8, −4.0; ESI-HRMS m/z calculated for C.sub.23H.sub.29N.sub.2O.sub.13P [M+Na].sup.+: 595.1299; Found 595.1305.

Synthesis of Compound 17

(74) To a stirred solution of compound 13 (100 mg, 0.24 mmol) in pyridine (2 mL) at room temperature POCl.sub.3 (0.024 mL, 0.26 mmol) was added and the mixture stirred. After 10 min, 4,5-dimethoxy-2-nitrobenzyl alcohol (153.4 mg, 0.72 mmol) was added and the reaction mixture was left stirring at the same temperature for 1 h. The reaction mixture was then concentrated in vacuo to yield crude product mixture, which after treatment with Dowex-H.sup.+ resin (50 mg) in methanol (2 mL) furnished compound 15 and 17. Filtration, concentration in vacuo and flash chromatography purification yielded compound 17 (55 mg, 48%) as a pure solid. (see FIG. 14)

Methyl 6-O-(4,5-dimethoxy-2-nitrobenzyloxyphosphoryl)-α-D-glucopyranoside 17

(75) R.sub.f 0.35 (1 water:2 isopropanol:4 ethyl acetate): [α].sub.D.sup.21+38.9 (c 0.64, MeOH), FT-IR (ATR) ν cm.sup.−1 3319 (br OH), 1521 (s, N═O), 1326 (s, N═O), 1220 (P═O); .sup.1H NMR (400 MHz, CD.sub.3OD): δ 7.74 (s, 1H, ArH), 7.50 (s, 1H, ArH), 5.33 (d, J=6.4 Hz, 2H, CH.sub.2Ar), 4.57 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 4.02 (s, 3H, OMe), 3.92 (s, 3H, OMe), 3.64-3.60 (m, 2H, H-6), 3.42 (dd, J.sub.5,4=9.6 Hz, J.sub.5,6b=2.8 Hz, 1H, H-5), 3.40 (brt, J.sub.3,2=9.2 Hz, J.sub.3,4=9.2 Hz, 1H, H-3), 3.39 (dd, J.sub.2,3=9.6 Hz, J.sub.2,1=3.0 Hz, 1H, H-2), 3.32 (s, 3H, OMe), 3.32-3.31 (m, 1H, H-4); .sup.13C NMR (400 MHz, CD.sub.3OD): δ 154.3, 147.9, 138.9, 131.1, 110.0, 107.9 (ArC), 100.0 (C-1), 73.6 (C-3), 72.2 (C-2), 71.3 (C-5), 70.0 (C-4), 64.8 (C-6), 64.5 (CH.sub.2Ar), 56.3 (OMe), 56.0 (OMe), 54.8 (OMe); .sup.31P NMR (162 MHz, CD.sub.3OD) δ 0.66; ESI-HRMS m/z calculated for C.sub.16H.sub.24NO.sub.13P [M−H].sup.−: 468.0907; Found 468.0905.

(76) The .sup.1H and .sup.13C NMR. Spectra of all compounds are shown in FIGS. 15-38.

(77) The signalling-precursor strategy was based on release by light (FIG. 2). Light-activated control is a potent strategy in biology because it can allow temporal and even spatial resolution that surpasses that of standard genetic methods (Mayer and Heckel, 2006, Angew Chem Int Ed Engl 45: 4900-4921). In principle, this resolution can be increased yet further when combined with small molecule chemical control given these too can be applied with localization and at predetermined time points (Adams and Tsien, 1993, Annu Rev Physiol 55: 755-784; Givens and Kueper, 1993, Chem. Rev. 93, 55-66; Ellis-Davies, 2007, Nat Methods 4: 619-628). The potency of such a method is increased further still when it leads to the release of a signalling molecule whose effect is amplified several fold. Notably, however, no such light-controlled approaches have, until now, been applied to sugar biology.

(78) Additionally, hydrophilic sugar molecules or charged molecules do not readily cross into plants unless actively transported. It was hypothesised that unnatural precursors could be designed that contain groups that would both mask charge/increase hydrophobicity and also be released by light. Four water-soluble precursors (1-4) of T6P were selected (FIG. 3). Each contained different light-sensitive moieties that functionally encapsulated T6P in an inactive and neutral form to facilitate entry into cells and that would then be liberated into active molecule upon irradiation with light: ortho-nitrobenzyl (oNB) in 1; 4,5-dimethoxynitrobenzyl (DMNB) in 2 and 4 and 2-(ortho-nitrophenyl)ethyl (oNPE) in 3. These differing groups were intended to permit the generation of create precursors with different behaviours in light and to fine-tune both uptake and release through change of both physical and chemical properties.

(79) Construction of the precursors (FIG. 3) used different phosphorus chemistries: phosphoramidite chemistry (Scheigetz and Roy, 2000, Synth. Commun. 30: 1437-1445; Arslan, et al., 1997, J. Am. Chem. Soc. 119: 10877-10887) to create P(III) intermediates that were then oxidized to corresponding P(V) phosphotriester intermediates or direct P(V) phosphorylation chemistry (FIG. 3). Regioselective access to the OH-6 group in trehalose was achieved through the use of trimethylsilyl (TMS) as a protecting group to form corresponding ethers. The TMS ether is chemically orthogonal to the phosphotriester group found in each of the light-sensitive moieties and its removal under mildly acidic conditions was successfully achieved. This was important since phosphate esters are highly prone to migration under basic conditions (Billington, 1989, Chem. Soc. Rev. 18: 83-122). Thus, intermediate 12 was prepared in gram quantities by regioselective removal of an 0-6 TMS ether group on persilylated trehalose (Ronnow et al., (1994) Carbohydr. Res. 260: 323-328). Overall phosphorylation of the revealed OH-6 hydroxyl in 12 involved reaction with phosphoramidites 9-11 (Scheigetz and Roy, 2000, Synth. Commun. 30: 1437-1445; Arslan, et al., 1997, J. Am. Chem. Soc. 119: 10877-10887) followed by in situ oxidation using tBuOOH. Using alternative direct P(V) chemistry a variant containing only a single DMNB was also created to explore the effect of different copy numbers of light-sensitive moieties; 12 was treated with 1 equiv. of POCl3 in pyridine (Meldal et al., (1992) Carbohydr. Res. 235: 115-127) followed by the addition of DMNB alcohol. Finally, all of the resulting intermediates were stirred in methanol in the presence of Dowex-H+ to induce mild deprotection which furnished the corresponding signalling precursors 1-4 (see SI). This synthetic route proved efficient and effective, allowing preparation of grams of signalling precursors at scales for application in plant trials (vide infra).

Example 2. Signalling Precursor Application to Water Stressed Wheat Plants

(80) Spring wheat (Triticum aestivum Cadenza) seeds were sown in Rothamsted Prescription Compost Mix (75% medium grade (L+P) peat, 12% screened sterilised loam, 3% medium grade vermiculite, 10% grit (lime-free), 3.5 kg Osmocote Exact 3-4 month/m.sup.3 (Scott's UK Professional, Ipswich, Suffolk, www.scottinternational.com), 0.5 kg PG mix/m.sup.3 (Hydro Agri Ltd., Immingham, UK), Wetting agent and Lime) and grown in controlled environment conditions with a photoperiod of 16 hours light, 8 hours dark, day/night temperatures of 20° C./16° C., photon flux density of 600 μmol m.sup.−2 s.sup.−1, and ambient relative humidity. Once the plants had reached Feekes stage 4, water was withheld for 10 days. On the 9th day, 30 ml 1 mM solutions of oNPE-T6P and DMNBT6P were applied to all above-ground biomass, on the 10th day the watering schedule was reinstated. Plants were harvested to measure biomass production every 5 days for 30 days after watering was reinstated. Both experiments were completed in replicates of 6.

(81) vi) Statistical Methods

(82) Analysis of Variance (ANOVA) was applied to data to test for differences between treatments. A natural log transformation was used where necessary to ensure constant variance. The GENSTAT statistical system was used for this analysis (2011, 14th edition, ©VSN International Ltd, Hemel Hempstead, UK).

(83) The effect of the signalling precursors upon plant resilience and recovery were tested. The results are shown in FIGS. 40-43. Drought is still the biggest global factor that limits crop yields, even in developed countries, such as the UK (Boyer, 1982, Science 218: 443-448). When 4-week-old wheat plants were sprayed with DMNB-T6P 2 or oNPE-T6P 3 (30 mL of 1 mM, once only) after 9 days of drought and then regrowth measured (recovery response) following resumption of watering 1 day after the initial chemical treatment, the effects were dramatic (FIGS. 40 and 41).

(84) In addition, regrowth (resurrection response) of new tissue from plants that had been cut back after drought treatment was higher in T6P precursor-treated plants (FIGS. 42 and 43). This demonstrates the capacity of T6P precursors to facilitate growth of new tissue (in the case of resurrection response) as well as salvage and grow new tissue (recovery response) in drought-treated plants. It should be noted that in all of these experiments, use of T6P solution alone gave identical results to use of water alone, consistent with the failure of T6P to enter into plants, further highlighting the success of the design principles for these signalling precursors.