1. Synthesis of Modified Substrates
for a Retaining Glycosyltransferase, Rv3032,
in MGLP Biosynthesis of Mycobacterium tuberculosis
Ziyan Zhao
ID: 25820729
Supervisor: Dr Seung Seo Lee
CHEM3034 BSc Research Project

2. Contents
• Research Background
• Project Overview
• Methods
• Results and Conclusion
• Future Perspectives
2

3. Research Background
• Mycobacterium tuberculosis remains a leading cause of death by
infectious disease, worldwide.
• M. tuberculosis synthesis unusual cytoplasm polymethylated poly-
saccharides (PMPs):
6-O-methylglucose lipopolysaccharides. (MGLP)
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Carbohydrates
Lipids
Proteins
Cell Envelop:
PMPs
Structure of Mycobacterium tuberculosis
Image from Wikipedia, http://en.wikipedia.org/wiki/File:Average_prokaryote_cell-_en.svg
Biosynthetic pathway of MGLP

4. Biosynthetic pathway of MGLP
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Acceptor Donor
Modified donor: prevent self-elongation of donor molecules
bind enzyme more efficiently
terminate elongation step

5. Biosynthetic pathway of MGLP
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Acceptor Donor
- Capture oxocarbenium ion-like transition state by kinetic analysis.
- Prove the proposed SNi mechanism for retaining glycotransferases.
a
b
R=Diglucosyl glycerate

6. Project Overview
• 4-Deoxy-4-fluoro-α-D-glucopyranoside
• 4-Deoxy-D-galactopyranose
6
Glucose derivative Modified
donor substrate
Glucose derivative Modified
donor substrate

7. Methods- Retrosynthesis
• 4-deoxy-4-fluoro-α-D-glucopyranoside
• 4-Deoxy-D-galactopyranose
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8. Methods- Forward synthesis
• 4-deoxy-4-fluoro-α-D-glucopyranoside
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Steps Method 1 Method 2
a BzCl, pyridine, RT, 3 days
b DAST, DCM, RT, Ar, overnight
c MeOH/H2O/Et3N, 110℃, Ar, overnight NaOMe, MeOH, RT, Ar, 18 hours
d Dowex 50 W-X8 H+, H2O, reflux, 25 hours MeSiCl3, NaI, CH3CN, Ar

9. Steps Method Steps Method
a , PTSA, DMF, RT, Ar, overnight d , toluene, 90 ℃, 18 h
b BnBr, NaH, DMF, RT, 18 h, overnight e n-Bu3SnH, AIBN, toluene, 90 ℃,
1.5 h
c TFA, Et3SiH, DCM, 0 ℃, 5 h f H2, Pd(OH)2/C, MeOH, RT,
overnight
Methods- Forward synthesis
• 4-Deoxy-D-galactopyranose
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10. Results and Conclusion
• Structure and purity proved by MS and NMR spectroscopy for each
step.
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Steps 1st
attempt
2nd
attempt
Lit.
Yield
Difficulties Improvements
Product1
a 20.61% 34.3% 65% Solidify product Mixed-solvent workups
b 12.7% 28.6% 41% TLC in DCM More polar eluent
c No
product
41.7% 78% Partial debenzoylation Stronger base
d No
product
Removal of methyl
group
Stronger demethylation
condition
Product2
a 26.4% 72%
b 14.6% 42.2% 83% Removal of DMF More extractions
c No
product
30% 63% Product identification;
reaction condition hard
to control
MS identification for TLC
spots; add reagents
dropwise.

11. Future Perspectives
• Continue synthesizing the two products.
• Add UDP onto the glucose derivatives by organic synthesis.
• Treat Rv3032 glycosyltransferase with modified substrates.
• Find evidence of intermediate by kinetic analysis methods.
• Prove the SNi mechanism for the chain elongation step.
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