This document summarizes a student's research project on synthesizing modified substrates for a glycosyltransferase enzyme involved in mycobacterium tuberculosis cell wall biosynthesis. The student aimed to synthesize 4-deoxy-4-fluoro-α-D-glucopyranoside and 4-Deoxy-D-galactopyranose derivatives to study the enzyme's reaction mechanism. Several steps were taken to synthesize each compound, with improvements made when initial attempts failed or yields were low. Future work involves completing the syntheses, adding UDP groups, testing the modified substrates with the enzyme, and analyzing reaction intermediates to validate the proposed mechanism.

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
4
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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