INDUCTION OF FLORAL DEVELOPMENT 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 induction of floral development in plants. The invention also concerns methods and the use of the compounds for the acceleration of floral development in treated compared with untreated plants.

Claims

1. A method of inducing flowering in a plant, wherein the method comprises treating the plant with a compound of formula (I), a phosphonate analogue thereof, or agriculturally acceptable salts thereof: ##STR00008## wherein: p is 0 or 1; R.sub.1 to R.sub.7 independently represent F, N.sub.3, NRR, C.sub.1-4 alkyl, (C.sub.1-4 alkyl)OH or OH, wherein R and R independently represent hydrogen or C.sub.1-4 alkyl; 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.

2. The method of claim 1, wherein the photo-labile protecting group is of formula (II): ##STR00009## wherein; (a) ring A represents an aryl or heterocyclic group; or (b) ring A represents a C.sub.6-10 aryl group or a 5- to 14-membered heterocyclic group containing one or more atoms selected from N, O and S, wherein the aryl or heterocyclic group is unsubstituted or substituted with one or more substituents selected from C.sub.1-4 alkyl, OR, halogen, CN, NRR, 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.1-4 alkyl, or wherein two adjacent substituents on the aryl or heterocyclic group together form a 5- or 6-membered heterocyclic ring containing one or more heteroatoms selected from N, O or S; or (c) ring A represents a phenyl, naphthalenyl or dibenzofuranyl ring; either (i) R.sub.10 and R.sub.11 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, 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.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; 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.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR 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): preferably wherein the photo-labile group is selected from: ##STR00010## wherein X represents the link to the remainder of the compound of formula (I).

3. (canceled)

4. The method of claim 1, wherein the photo-labile protecting group is of formula (III): ##STR00011## wherein either Z represents N, Y represents CR.sub.36 and Z and Y are linked by a double bond; or Z represents O, Y represents CO and Z and Y are linked by a single bond; R.sub.36 represents CR.sub.37R.sub.38X; when Y represents CR.sub.36, R.sub.35 represents hydrogen, and when Y represents CO, R.sub.35 represents CR.sub.37R.sub.38X; either (i) R.sub.37 and R.sub.38 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, or (ii) two R.sub.37 groups on adjacent photolabile protecting groups together form a bond and R.sub.38 represents hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; R.sub.32 represents OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4 alkyl; and R.sub.31, R.sub.33 and R.sub.34 are independently selected from hydrogen, halogen, OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4alkyl; wherein X represents the link to the remainder of the compound of formula (I) preferably wherein the photo-labile protecting group is selected from: ##STR00012##

5. (canceled)

6. (canceled)

7. (canceled)

8. The method of claim 1, wherein: (a) the photo-labile protecting group is of formula (IIa): ##STR00013## wherein; ring A represents an unsubstituted or substituted group selected from phenyl, naphthyl or dibenzofuranyl, wherein a substituted phenyl, naphthyl or dibenzofuranyl group is a phenyl, naphthyl or dibenzofuranyl group having one or two methoxy substituents, or a phenyl, naphthyl or dibenzofuranyl group wherein two adjacent ring positions are substituted with a CH.sub.2OCH.sub.2 moiety; and R.sub.10 represents hydrogen, methyl, CF3 or COOH; wherein X represents the link to the remainder of the compound of formula (I) or (b) the photolabile protecting group is of formula (IIIa): ##STR00014## wherein: R.sub.32 represents OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; and R.sub.33 represents hydrogen, Br, OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; wherein X represents the link to the remainder of the compound of formula (I); preferably wherein: a) R.sub.33 represents H and R.sub.32 represents OMe, NMe.sub.2, NEt.sub.2 or OCH.sub.2COOH; or b) R.sub.33 represents Br and R.sub.32 represents OH; or c) R.sub.33 and R.sub.32 both represent OCH.sub.2COOH.

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. The method of claim 1, wherein the plant is a dicotyledonous plant: preferably wherein the plant is a Brassica plant.

14. (canceled)

15. (canceled)

16. A method of accelerating the floral development of a plant, wherein the method comprises treating the plant with a compound of formula (I), a phosphonate analogue thereof, or agriculturally acceptable salts thereof: ##STR00015## wherein: p is 0 or 1; R.sub.1 to R.sub.7 independently represent F, N.sub.3, NRR, C.sub.1-4 alkyl, (C.sub.1-4 alkyl)OH or OH, wherein R and R independently represent hydrogen or C.sub.1-4 alkyl; 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.

17. The method of claim 1, wherein the method comprises treating the plant in a vegetative growth phase.

18. The method of claim 1, wherein the compound is applied together with at least one fertilizer, fungicide, herbicide, insecticide or plant growth regulator.

19. (canceled)

20. (canceled)

21. The method of claim 1, wherein the plant has a greater number of floral buds compared with untreated plants.

22. A method of co-ordinating the floral development of plants, comprising treating the growing plants with a compound as of formula (I), a phosphonate analogue thereof, or agriculturally acceptable salts thereof: ##STR00016## wherein: p is 0 or 1; R.sub.1 to R.sub.7 independently represent F, N.sub.3, NRR, 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.

23. The method of claim 16, wherein the photo-labile protecting group is of formula (II): ##STR00017## wherein; (a) ring A represents an aryl or heterocyclic group; or (b) ring A represents a C.sub.6-10 aryl group or a 5- to 14-membered heterocyclic group containing one or more atoms selected from N, O and S, wherein the aryl or heterocyclic group is unsubstituted or substituted with one or more substituents selected from C.sub.1-4 alkyl, OR, halogen, CN, NRR, 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.1-4 alkyl, or wherein two adjacent substituents on the aryl or heterocyclic group together form a 5- or 6-membered heterocyclic ring containing one or more heteroatoms selected from N, O or S; or (c) ring A represents a phenyl, naphthalenyl or dibenzofuranyl ring; either (i) R.sub.10 and R.sub.11 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, 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, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; 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.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR 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); preferably wherein the photo-labile group is selected from: ##STR00018## wherein X represents the link to the remainder of the compound of formula (I).

24. The method of claim 16, wherein the photo-labile protecting group is of formula (III): ##STR00019## wherein: either Z represents N, Y represents CR.sub.36 and Z and Y are linked by a double bond; or Z represents O, Y represents CO and Z and Y are linked by a single bond; R.sub.36 represents CR.sub.37R.sub.38X; when Y represents CR.sub.36, R.sub.35 represents hydrogen, and when Y represents CO, R.sub.35 represents CR.sub.37R.sub.38X; either (i) R.sub.37 and R.sub.38 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms; OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, or (ii) two R.sub.37 groups on adjacent photo-labile protecting groups together form a bond and R.sub.38 represents hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; R.sub.32 represents OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4 alkyl; and R.sub.31, R.sub.33 and R.sub.34 are independently selected from hydrogen, halogen, OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4alkyl; wherein X represents the link to the remainder of the compound of formula (I); preferably wherein the photo-labile protecting group is selected from: ##STR00020##

25. The method of claim 16, wherein: (a) the photo-labile protecting group is of formula (IIa): ##STR00021## wherein; ring A represents an unsubstituted or substituted group selected from phenyl, naphthyl or dibenzofuranyl, wherein a substituted phenyl, naphthyl or dibenzofuranyl group is a phenyl, naphthyl or dibenzofuranyl group having one or two methoxy substituents, or a phenyl, naphthyl or dibenzofuranyl group wherein two adjacent ring positions are substituted with a CH.sub.2OCH.sub.2-moiety; and R.sub.10 represents hydrogen, methyl, CF3 or COOH; wherein X represents the link to the remainder of the compound of formula (I) or (b) the photolabile protecting group is of formula (IIIa): ##STR00022## wherein; R.sub.32 represents OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; and R.sub.33 represents hydrogen, Br, OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; wherein X represents the link to the remainder of the compound of formula (I); preferably wherein: a) R.sub.33 represents H and R.sub.32 represents OMe, NMe.sub.2, NEt.sub.2 or OCH.sub.2COOH; or b) R.sub.33 represents Br and R.sub.32 represents OH; or c) R.sub.33 and R.sub.32 both represent OCH.sub.2COOH.

26. The method as claimed in claim 16, wherein the method comprises treating the plant in a vegetative growth phase and/or the compound is applied together with at least one fertilizer, fungicide, herbicide, insecticide or plant growth regulator.

27. The method of claim 16, wherein the plant has a greater number of floral buds compared with untreated plants.

28. The method of claim 22, wherein the photo-labile protecting group is of formula (II): ##STR00023## wherein: (a) ring A represents an aryl or heterocyclic group; or (b) ring A represents a C.sub.6-10 aryl group or a 5- to 14-membered heterocyclic group containing one or more atoms selected from N, O and S, wherein the aryl or heterocyclic group is unsubstituted or substituted with one or more substituents selected from C.sub.1-4 alkyl, OR, halogen, CN, NRR, 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.1-4 alkyl, or wherein two adjacent substituents on the aryl or heterocyclic group together form a 5- or 6-membered heterocyclic ring containing one or more heteroatoms selected from N, O or S; or (c) ring A represents a phenyl, naphthalenyl or dibenzofuranyl ring; either (i) R.sub.10 and R.sub.11 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, 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, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; 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.1-4 alkyl which is unsubstituted or substitute with one or more halogen atoms, OR, halogen, NRR 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); preferably wherein the photo-labile group is selected from: ##STR00024## wherein X represents the link to the remainder of the compound of formula (I).

29. The method of claim 22, wherein the photo-labile protecting group is of formula (III): ##STR00025## wherein; either Z represents N, Y represents CR.sub.36 and Z and Y are linked by a double bond; or R.sub.36 represents CR.sub.37R.sub.38X; when Y represents CR.sub.36, R.sub.35 represents hydrogen, and when Y represents CO, R.sub.35 represents CR.sub.37R.sub.38X; either (i) R.sub.37 and R.sub.38 are the same or different and are selected from hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms; OR, halogen, NRR or CO.sub.2R, wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl, or (ii) two R.sub.37 groups on adjacent photolabile protecting groups together form a bond and R.sub.38 represents hydrogen, C.sub.1-4 alkyl which is unsubstituted or substituted with one or more halogen atoms, OR, halogen, NRR or CO.sub.2R wherein R and R are independently selected from hydrogen and C.sub.1-4 alkyl; R.sub.32 represents OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4 alkyl; and R.sub.31, R.sub.33 and R.sub.34 are independently selected from hydrogen, halogen, OR, NRR, O(C.sub.1-4alkyl)-COOR, O(C.sub.1-4alkyl)-OR or O(C.sub.1-4alkyl)-NRR, wherein R and R independently represent hydrogen or C.sub.1-4alkyl; wherein X represents the link to the remainder of the compound of formula (I); preferably wherein the photo-labile protecting group is selected from: ##STR00026##

30. The method of claim 22, wherein: (a) the photo-labile protecting group is of formula (IIa): ##STR00027## wherein; ring A represents an unsubstituted or substituted group selected from phenyl, naphthyl or dibenzofuranyl, wherein a substituted phenyl, naphthyl or dibenzofuranyl group is a phenyl, naphthyl or dibenzofuranyl group having one or two methoxy substituents, or a phenyl, naphthyl or dibenzofuranyl group wherein two adjacent ring positions are substituted with a CH.sub.2OCH.sub.2-moiety; and R.sub.10 represents hydrogen, methyl, CF3 or COOH; wherein X represents the link to the remainder of the compound of formula (I); or (b) the photo-labile protecting group is of formula (IIIa): ##STR00028## wherein; R.sub.32 represents OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; and R.sub.33 represents hydrogen, Br, OR, NRR or O(C.sub.1-4alkyl)-COOR, wherein R and R independently represent hydrogen or C.sub.1-2 alkyl; wherein X represents the link to the remainder of the compound of formula (I); preferably wherein: a) R.sub.33 represents H and R.sub.32 represents OMe, NMe.sub.2, NEt.sub.2 or OCH.sub.2COOH; or b) R.sub.33 represents Br and R.sub.32 represents OH; or c) R.sub.33 and R.sub.32 both represent OCH.sub.2COOH.

31. The method as claimed in claim 22, wherein the method comprises treating the plant in a vegetative growth phase and/or the compound is applied together with at least one fertilizer, fungicide, herbicide, insecticide or plant growth regulator.

32. The method of claim 22, wherein the plant has a greater number of floral buds compared with untreated plants.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The invention will now be described in detail with reference to Examples and with reference to the accompanying drawings, in which:

[0051] FIG. 1 shows a schematic diagram of a chemical strategy to control trehalose-6-phosphate (T6P) in plants.

[0052] FIG. 2 shows a schematic diagram illustrating the principle of photo-activated release of T6P in planta from plant permeable signalling precursors of T6P.

[0053] FIG. 3 shows the synthesis of bis-(2-nitrobenzyl)-N,N-dilsopropylphosphoramidite 9.

[0054] FIG. 4 shows the synthesis of bis-(4,5-dimethoxy-2-nitrobenzyl)-N,N dilsopropylphosphoramidite 10.

[0055] FIG. 5 shows the synthesis of bis-[1-(2-nitrophenyl)-ethyl]-N,N-diisopropylphosphoramidite 11.

[0056] FIG. 6 shows the synthesis of compound 1.

[0057] FIG. 7 shows the synthesis of compound 2.

[0058] FIG. 8 shows the synthesis of compound 3.

[0059] FIG. 9 shows the synthesis of compound 4.

[0060] FIG. 10 shows the synthesis of compound 13.

[0061] FIG. 11 shows the synthesis of compound 14.

[0062] FIG. 12 shows the synthesis of compound 15.

[0063] FIG. 13 shows the synthesis of compound 16.

[0064] FIG. 14 shows the synthesis of compound 17.

[0065] FIG. 15 shows the .sup.1H and .sup.13C NMR Spectra of compound 9.

[0066] FIG. 16 shows the .sup.1H and .sup.13C NMR Spectra of compound 9.

[0067] FIG. 17 shows the .sup.1H and .sup.13C NMR Spectra of compound 10.

[0068] FIG. 18 shows the .sup.1H and .sup.13C NMR Spectra of compound 10.

[0069] FIG. 19 shows the .sup.1H and .sup.13C NMR Spectra of compound 11.

[0070] FIG. 20 shows the .sup.1H and .sup.13C NMR Spectra of compound 11.

[0071] FIG. 21 shows the .sup.1H and .sup.13C NMR Spectra of compound 1.

[0072] FIG. 22 shows the .sup.1H and .sup.13C NMR Spectra of compound 1.

[0073] FIG. 23 shows the .sup.1H and .sup.13C NMR Spectra of compound 2.

[0074] FIG. 24 shows the .sup.1H and .sup.13C NMR Spectra of compound 2.

[0075] FIG. 25 shows the .sup.1H and .sup.13C NMR Spectra of compound 3.

[0076] FIG. 26 shows the .sup.1H and .sup.13C NMR Spectra of compound 3.

[0077] FIG. 27 shows the .sup.1H and .sup.13C NMR Spectra of compound 4.

[0078] FIG. 28 shows the .sup.1H and .sup.3C NMR Spectra of compound 4.

[0079] FIG. 29 shows the .sup.1H and .sup.13C NMR Spectra of compound 14.

[0080] FIG. 30 shows the .sup.1H and .sup.13C NMR Spectra of compound 14.

[0081] FIG. 31 shows the .sup.1H and .sup.13C NMR Spectra of compound 15.

[0082] FIG. 32 shows the .sup.1H and .sup.13C NMR Spectra of compound 15.

[0083] FIG. 33 shows the .sup.1H and .sup.13C NMR Spectra of compound 16.

[0084] FIG. 34 shows the .sup.1H and .sup.13C NMR Spectra of compound 16.

[0085] FIG. 35 shows the .sup.1H and .sup.13C NMR Spectra of compound 17.

[0086] FIG. 36 shows the .sup.1H and .sup.13C NMR Spectra of compound 17.

[0087] FIG. 37 shows the .sup.1H and .sup.13C NMR Spectra of compound T6P.

[0088] FIG. 38 shows the .sup.1H and .sup.13C NMR Spectra of compound T6P.

[0089] FIG. 39 shows the known biochemical structures and one-pot synthesis of designed permeable, signalling precursor variants from suitable precursors.

[0090] FIG. 40 shows the effect of spraying T6P signalling precursors on the height of the flowering inflorescence measured at 26 days after sowing (DAS). Panel A shows Col-0 plant and Panel B shows tps7 plants. Right hand bars in each panel represent plants treated with 1 mM oNPE. Left hand bars in each panel represent plants treated with water (controls). Treatment with 1 mM oNPE in Col-0 and tps7 resulted in taller inflorescences. This reflects the advancement of flowering by oNPE.

[0091] FIG. 41 shows a possible role of T6P and tps7 in control of flowering in plants and crops.

DETAILED DESCRIPTION

Examples

[0092] 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 TSP

[0093] T6P is plant impermeable (FIG. 2). In order to alter TOP levels in planta, plant permeable signalling precursor variants were designed and synthesised in a single pot reaction starting from suitable precursors.

Synthesis of Signalling-Precursor Compounds 1-4

[0094] 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.

[0095] 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.

[0096] 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

[0097] 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

[0098] Mp 71-72 C. [lit..sup.5 Mp 71-73 C.] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.10 (d, J=8.4 Hz, 2H, H-3 and H-3), 7.86 (d, J=8.0 Hz, 2H, H-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, 2CH(CH.sub.3).sub.2), 1.25 (d, J==8.5 Hz, 12H, 2CH(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.2).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) 149.0; ESI-LRMS nm/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

[0099] 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).

[0100] 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

[0101] .sup.1H NMR (400 MHz, CDCl.sub.3) 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, 2OMe), 3.94 (s, 6H, 2OMe), 3.85-3.70 (m, 2H, 2CH(CH.sub.3).sub.2) 1.27 (d, J=8.5 Hz, 12H 2CH(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)), 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

[0102] 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.6 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

[0103] Isolated as a dia-stereomeric mixture. .sup.1H NMR (400 MHz, CDCl.sub.3) 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, 2CH(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); .sup.13C NMR (100 MHz, CDCl.sub.3) 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-5S), 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+H].sup.+; 464.1; Found 464.1.

Synthesis of Compound 1

[0104] 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 0.4M solution in CH.sub.3CN) in anhydrous CH.sub.2Cl.sub.2 (4 mL) under an argon atmosphere at 0 C. m bis-(2-nitrobenzyl)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 equiv0.1 mL of 5M 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

[0105] R.sub.f0.60 (1 water; 2 isopropanol: 4 ethyl acetate). [].sub.D.sup.21+80.6 (c1.0, MeOH); FT-IR (ATR) v cm.sup.1 (3347 (br, OH), 1526 (s, NO), 1343 (s, NO), 1255 (PO); .sup.1H NMR (500 MHz, D.sub.2O) 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.r,z=3.6 Hz, 1H, H-1), 4.93 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 4.40 (dd, J.sub.6a,6b=11.0 Hz, J.sub.6a,5=2.0 Hz, 1H, H-6a), 4.35 (dd, J.sub.6n,6a=11.0 Hz, J.sub.6b,5=4.5 Hz, 1H, H-6b), 3.93 (td, J.sub.54=10.0 Hz and J.sub.5,6a=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.23=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.43=9.6 Hz, J.sub.45=9.6 Hz, 1H, H-4), 3.22 (t, J.sub.4,3=9.6 Hz J.sub.4,59.6 Hz, 1H, H-4); .sup.13C NMR (125 MHz, D20) 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 ((H.sub.12Ar), 61.6 (C-6); .sup.31P NMR (162 MHz, D.sub.2O) 0.11; ESI-HRMS m/z calculated for C.sub.26H.sub.33N.sub.2O.sub.1KP [M+Na].sup.+ 715.1368; Found 715.1368.

Synthesis of Compound 2

[0106] 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 5M 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

[0107] R.sub.f 0.50 (1 water; 2 isopropanol: 4 ethyl acetate); [].sub.D.sup.21+64.8 (c1.1, MeOH); FT-IR (ATR) v cm.sup.1 3347 (br, OH), 1519 (s, NO), 1326 (s, NO), 1220 (P=O): .sup.1H NMR (500 MHz, CD.sub.3OD) 7.53 (s, 2H, ArH), 7.03 (s, 2H, ArH), 5.37 (d, J=8.0 Hz, 4H, 2CH2Ar), 4.95 (d, J.sub.12=4.0 Hz, 1H, H-1), 4.91 (d, J.sub.1,2=4.0 Hz, 1H, H-1), 4.30 (dd, J.sub.6a,6b=11.6 Hz, J.sub.6a,6a=2.0 Hz, 1H, H-6a), 4.35 (dd, J.sub.6b,6a=11.0 Hz, J.sub.6b,5=3.6 Hz, 1H, H-6b), 3.94 (td, J.sub.5,4=10.0 Hz and J.sub.5,6a=2.0 Hz, 1H, H-5), 3.71 (t, J.sub.32=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.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=11.6 Hz and J.sub.6b,5=5.6 Hz, 1H, H-6b), 3.35 (dd, J.sub.3,3=8.4 Hz, J.sub.2,=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.4,5=8.8 Hz, J.sub.45=8.8 Hz, 1H, H-4), 3.24 (t, J.sub.4,3=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), 10.4 (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 (2OMe), 55.8 (2OMe); .sup.31P NMR (162 MHz CD.sub.3OD) 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

[0108] 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., his-[i-(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 or starting material (18 h), tBuOOH (32.5 mg. 0.36 mmol, 3.0 equiv.0.1 mL of 5M 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

[0109] Isolated as mixture of four diastereomers. R.sub.f0.65 (1 water:2 isopropanol:4 ethyl acetate); FT-IR (ATR) v cm.sup.1 3394 (br, OH), 1521 (s, NO), 1326 (s, NO), 1276 (P=O); .sup.1H NMR (500 MHz, CD.sub.3OD) 7.91-7.05 (m, 8H, ArH), 5.92-5.84 (m, 2H, 2CHMe), 5.04-4.94 (m, 2H, H-1 and H-1), 3.90-3.60 (m, 7H), 3.41-3.05 (m, 5H), 1.56-1.46 (m, 6H, 2CHMe); .sup.13C NMR (125 MHz, CD.sub.3OD 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) 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 [M+Na].sup.+; 743.1677; Found 743.1676.

Synthesis of Compound 4

[0110] 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)-trehalose 4

[0111] R.sub.f 0.33 (1 water:2 isopropanol:4 ethyl acetate); [].sub.D.sup.21+48.7 (c1.1, MeOH); FT-IR (ATR) v cm.sup.1 3312 (br, OH), 1521 (s, NO), 1326 (s, NO), 1220 (P=O); .sup.1H NMR (500 MHz, CD.sub.3OD) 7.62 (s, 1H, ArH), 7.39 (s, 1H, ArH), 5.21 (d, J=6.0 Hz, 2H, CH.sub.2A), 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, J.sub.2,3=7.2 Hz and J.sub.2,1=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) 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) 2.18 (P=O): ESI-HRMS m/z calculated for C.sub.21H.sub.32NO.sub.18P [MH].sup.: 616.1279; Found 616.1273.

Synthesis of Compound 13

[0112] Methyl tetra-O-trimethylsiyl--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 (315 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--D-glucopyranoside 13

[0113] colourless solid [].sub.D.sup.21+95.3 (c 1, CHCl.sub.3), [lit.sub.2 [].sub.D.sup.21+93 (c3, CHCl.sub.3)]; .sup.1H NMR (400 MHz, CDCl.sub.3): 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); 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.36O.sub.6Si.sub.3 [M+Na].sup.+: 433.18; Found 433.20.

Synthesis of Compound 14

[0114] 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 0.4M 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)--D-glucopyranoside 14

[0115] R.sub.f 0.50 (1 Methanol: 9 dichloromethane): [].sub.D.sup.21+49.5 (c1.0, MeOH); FT-IR (ATR) v cm.sup.1 3354 (br, OH), 1525 (s, NO), 1342 (s, NO), 1255 (P=O); .sup.1H NMR (400 MHz, CD.sub.3OD): 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, 2CH2Ar), 4.50 (d, J.sub.1,2=3.6 Hz, 1H, H-1), 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.6a,6b=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): 147.3 (qC Ar), 143.3 (qC Ar), 134.2, 132.0, 129.4, 128.8, 125.0 (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),

[0116] 54.7 (OMe); .sup.31P NMR (162 MHz, CD.sub.3OD) 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

[0117] 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 0.4M soln in CH.sub.3CN) in dry CH.sub.2Cl.sub.2 (8 mL) under an argon atmosphere at 0C, 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--D-glucopyranoside 15

[0118] R.sub.f0.40 (1 Methanol: 9 dichloromethane); [].sub.D.sup.21+40.7 (c1.09, MeOH); FT-IR (ATR) v cm.sup.13355 (br, OH), 1519 (s, NO), 1326 (s, NO), 1220 (P=O); .sup.1H NMR (400 MHz, CD.sub.3OD): 7.51 (s, 2H, ArH), 7.03 (s, 2H, ArH), 5.37 (d, J=8.0 Hz, 4H, 2CH.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.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.5,6b=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): 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) 1.62; ESI-HRMS m/z calculated for C2H.sub.33N.sub.2O.sub.17P [M+Na].sup.+: 687.1409; Found 687, 1421.

Synthesis of Compound 16

[0119] 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 0.4M soln in CH.sub.3CN) in dry CH2Cl.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 equiv0.2 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]--D-glucopyranoside 16

[0120] R.sub.f 0.55 (1 Methanol:9 dichloromethane); Isolated as a mixture of four diastereomers, FT-IR (ATR) v 3334 cm.sup.1 (br, OH), 1520 (s, NO), 1325 (s, NO), 1219 (P=O); .sup.1H NMR (400 MHz, CD.sub.3OD); 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, 2CH(CH.sub.3), 4.57-4.45 (4d, J.sub.1=3.6 Hz, 1H, H-1), 4.16-3.89 (m, 2H, H-6). 3.51-3.40 (m, 2H, H-5 and H-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, 2CH(CH.sub.3): .sup.13C NMR (400 MHz, CD.sub.3OD): 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) 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

[0121] 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)--D-glucopyranoside 17

[0122] R.sub.f 0.35 (1 water: 2 isopropanol: 4 ethyl acetate): [].sub.D.sup.21+38.9 (c0.64. MeOH), FT-IR (ATR) v cm.sup.1 3319 (br OH), 1521 (s, NO), 1326 (s, NO), 1220 (P=O); .sup.1H NMR (400 MHz, CD.sub.3OD): 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): 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) 0.66; ESI-HRMS m/z calculated for C.sub.16H.sub.24NO.sub.33P [MH].sup.: 468.0907; Found 468.0905.

[0123] The .sup.1H and .sup.13C NMR Spectra of all compounds are shown in FIGS. 15-38.

[0124] 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 Engi 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.

[0125] Additionally, hydrophilic sugar molecules or charged molecules do not readily cross into plants unless actively transported. It was hypothesized 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.

[0126] 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 trimethytsilyl (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. Ectopic Application of TSP Chemicals Controls Flowering Time in Arabidopsis Plants

[0127] To demonstrate efficacy of T6P chemicals to regulate flowering, Arabidopsis thaliana wild type and a delayed flowering mutant (attps7) have been used. In both cases it is possible to advance the development of the flowering inflorescence by several centimetres and advance flowering time by up to a week.

Plant Growth Conditions

[0128] Seeds of Arabidopsis thaliana, ecotype Columbia (Col-0) and attps7 knockout mutants were surface-sterilized with 70% of ethanol for 2 min and 75% household bleach for 7 min, and grown on nutrient agar media (0.5Murashige and Skoog plus Gamborg's vitamins (Sigma). 0.5% Sucrose and 0.7% Agar) in Petri plates. Seeds on the Petri plates were placed in a growth chamber (22 C., 150 mol m.sup.2 s.sup.1, 16-h day). Seven days after plating, seedlings were transferred to pots of soil containing Rothamsted standard compost mix and full nutrition (one seedling per pot). Forty plants per A. thaliana type (n=40 Col-0, n=40 TPS7_3, n=40 TPS7_4.15) were grown at 22CC, 250 mol m.sup.2 s.sup.1 irradiance, 16-h day. Eight days after sowing (DAS) plants were sprayed with 0.5 ml 1 mM oNPE-T6P (ortho-nitrphenyl)ethyl trehalose 6-phosphate dissolved in distilled water with the addition of 0.1% of Tween20 during the middle of the mid-photoperiod. Twenty-four hours after spraying (9 DAS), plants sprayed with 1 mM oNPE-T6P were exposed to 600 mol m.sup.2 s.sup.1 irradiance for 1 h to release T6P from oNPE-T6P. Spraying was repeated at 12 and 16 DAS.

[0129] First, the appearance of the flower bud was quicker in plants sprayed with oNPE-T6P than with water.

TABLE-US-00001 TABLE 1 The number of flower buds that had appeared at day 5 after spraying in Col-0 and tps7 mutants. In all cases more flower buds had appeared in oNPE-treated plants compared to control shown as crosses in highlighted boxes (100% in Col-0, 90% and 90% in tps7 mutants compared to 70%, 70% and 30% in Col-0, and tps7 mutants treated with water only. Water (control) 1 mM oNPE-T6P Day 5 Day 5 N Col-0 TPS7_3 TPS7_4.15 Col-0 TPS7_3 TPS7_4.15 1 X X X X X 2 X X X X X 3 X X X X 4 X X X X 5 X X X X X 6 X X X X 7 X X X X X 8 X X X X 9 X X X X X X 10 X X X

[0130] Height of the flowering inflorescence was then measured at 26 DAS. Treatment with 1 mM oNPE in Col-0 and tps7 resulted in taller inflorescences (right hand bars in each case compared to left hand bars which represent water controls) (FIG. 40). This reflects the advancement of flowering by oNPE.

[0131] T6P chemicals therefore represent a novel way to control flowering time in plants.

[0132] The molecular basis of the signalling pathway involved in transducing the T6P sugar signal for flowering is not well understood. Without wishing to be bound by any particular theory, the generic nature of T6P signalling in plants and crops indicates that T6P is a universal mechanism for the control of flowering time. A possible role of T6P and tps7 in control of flowering in plants and crops is illustrated in FIG. 41.

[0133] T6P chemicals therefore provide a direct method to control flowering time without the need for complex genetics. Flowering time, flowering time coordination and repeat flowering can be controlled with the T6P chemicals.