Mission Statement 

To discover and develop a new class of pharmaceutical compounds based on the biological functions of the interactions between glycoconjugates and their receptors.

Currently, GlycoTech is involved in a corporate alliance with Novartis, Basel Switzerland. Our objective is to develop novel compounds to inhibit the interaction of the selectins with their carbohydrate ligands for the treatment of disease (eg. inflammation) or conditions (eg. reperfusion injury) caused by the recruitment of leukocytes to affected tissues.

GlycoTech also maintains a collaborative research agreement with the Chicago Institute for Neurosurgery and Neuroresearch to study the role of carbohydrates in the functioning of the central nervous system. Applications from this research involve novel therapeutic approaches for the treatment of brain tumors, Alzheimer's disease, and neurite regeneration.

Many tumor markers defined by monoclonal antibodies have been identified as carbohydrate structures. GlycoTech has developed an extensive data base of monoclonal antibodies directed against carbohydrates and is aggressively pursuing their use in identifying early stages of cancer with several diagnostic companies.

GlycoTech is actively seeking other pharmaceutical partners and offers a combined range of expertise focusing on the Glycobiology area. The corporate structure and strategy of GlycoTech allows its partners to quickly obtain this unique expertise and avoid the obligations encountered with the fixed financial responsiblities associated with internal organic growth. We welcome the opportunity to meet with all interested parties to explore mutually constructive collaborations.

 

FINE CHEMICALS and REAGENTS BUSINESS

The purpose of this division of GlycoTech is to make techniques and reagents available to all scientists in order to broaden investigations of carbohydrates into other fields of research such as Cell Biology, Immunology, Neurobiology, and Molecular Biology. The first section of the catalog contains some useful protocols written by well known scientists covering a wide range of topics in Glycobiology. The following sections contain a variety of unique carbohydrate reagents that allow many of these protocols to be followed in laboratories not necessarily specializing in Glycobiology. This catalog also welcomes the participation of scientists within this field with the objective to actively support their research and discoveries. GlycoTech's long-term goal is to use our technology and reagents for research towards new discoveries of the biological functions of carbohydrates based on the interaction of glycoconjugates and their receptors. 

Glycobiology Research and Training 

Composition Analysis by Alditol Acetates

​MATERIALS

• 4 N Trifluoroacetic Acid (TFA)

• Acetic Anhydride (Sigma, 539996)

• Pyridine (Sigma, 270970)

• NaBH4 (Sigma, 213462) or NaBD4 (Sigma, 205591)

• 1 M NH4OH

• Myo-Inositol (Sigma, 57569)

• 50% Iso-propyl alcohol (IPA)

• 1 mg/mL solution of standard sugars used for alditol acetates (AA): Rhamnose, Fucose, Ribose,

Arabinose, Xylose, Mannose, Galactose, Glucose, N-acetyl Glucosamine, N-acetyl

Galactosamine, N-acetyl Mannosamine.

Standard Preparation:​

• To prepare an alditol acetate standard place 2 µg of neutral and amino sugars, into a sample tube

along with 1 µg of myo-inositol as internal standard

Sample Preparation:

• 50-100 µg of sample are taken in a screw cap tube and 1 µg of myo-inositol is added to it as

internal standard. The samples can also be in known volume of water.

PROCEDURE

1. Add an equal volume of 4 N TFA to the standard and samples to make a final concentration of

2 N TFA and hydrolyze at 100 o C for 4 hrs

2. Cool samples to room temperature and centrifuge at 2000 rpm for 2 min to bring the

condensation down to the bottom of the tube.

3. Remove the TFA using dry nitrogen flush on an evaporation heat block. Use slight heat ~37

o C to fasten the evaporation.

4. Add 100 µL of 50% IPA and re-evaporate the sample. Repeat the IPA wash once more to

fully remove TFA. Repeating this step twice will remove the residual acid completely,

however to ensure complete removal of the acid check the pH with a pH paper.

5. Prepare the reducing agent by dissolving 10 mg of NaBH4 in 1 mL of 1M ammonium

hydroxide. Add ~100-150 µL of reducing agent to each sample and mix by vortexing.

6. Incubate samples at room temperature for 16h or overnight. Make sure to loosen the cap as

this reaction forms hydrogen gas.

7. Check the pH of the reduced sample with pH paper, it should be alkaline (pH>12). Cool down

the sample on ice-water bath for 1 min then add ice-cold 30% aqueous acetic acid drop wise

to neutralize the sample. Vortex thoroughly after addition of each drop of acid and return the

sample to the ice-water bath frequently throughout this process. Complete neutralization

occurs when there is no effervescence after acid is added. To ensure neutralization use pH

paper to check the pH.

8. Dry the neutralized samples by dry nitrogen flush, slight heat (<37 o

C) can be applied to

fasten the evaporation however don’t use heat when handling permethylated sample). The

dried sample may look like syrupy or like a gel. At this stage add 100 µL of Methanol, vortex

thoroughly to completely dissolve the sample and again evaporate the sample by dry nitrogen

flush, repeat for a total of three times.

Note: Boric acid formed by neutralization is removed as volatile methyl borate, this step is

necessary as excess boric acid interferes with acetylation of sugars.

9. Next add 100 µL of 9:1 methanol:glacial acetic acid to the dried sample and vortex to dissolve

completely. Re-evaporate using dry nitrogen flush. Repeat this step at least three times

followed by evaporating the samples using absolute methanol (3 times, 100 µL each time).

The sample should form a white crust of sodium acetate around the wall of the glass tube.

10. Place the dried samples in a vacuum desiccator over P2O5 for 2-3 hours.

11. To acetylate the sample add 50 µL of pyridine and 50 µL of acetic anhydride. Vortex to mix

the reagents and sonicate (30-40 sec) to break the solid sticking on the wall of the tube. Heat

the reaction mixture at 100 o

C for 1 hour with vortexing at 20 min intervals.

12. Cool the samples to room temperature and then centrifuge at 2000 rpm for 2 min to bring the

condensation down to the bottom of the tube. Remove pyridine and acetic anhydride using

dry nitrogen flush on the evaporator (DO NOT APPLY HEAT at this stage as the samples are

highly volatile). After the samples are dried down completely add 100 µL of toluene, vortex

and evaporate toluene using dry nitrogen flush.

Note: Toluene is added to remove trace of pyridine and acetic anhydride which prevents peak

trailing in the GC-MS spectrum.

13. Dissolve the dried samples in 1 mL of dicholormethane and sonicate to break down the solid

crust. Vortex the reaction mixture and centrifuged at 2000 rpm for 2 min at 15 o

C. The crust should be settled down at the bottom,

14. Filter the supernatant through a Pasteur pipette filled with glass wool. Pre-wash the glass

wool with a small amount of dichloromethane.

15. Collect the filtrate and remove the dichloromethane by dry nitrogen flush.

16. Reconstitute the samples in 100-150 µL of dichloromethane and transfer into a sample vial for

analysis by GC-MS.

GC-MS SETTINGS FOR ALDITOL ACETATES:

Carrier Gas: Helium

Inlet Conditions:

• Temperature: 220 o C

• Pressure: 11.649 psi

• Flow: 22.24 mL/min

1. GC-MS Transfer Line Temp: 280 o C
2. MS Source: 230 oC
3. MS Quad: 150 o C
4. Column: DB-5 or Equivalent, 30 m x 0.25 mm x 0.25 µm
5. Column Flow: 1.1971 mL/min
6. Injection Volume: 1 µL
7. Run Time 48 min
8. Conditions oC/min o C Hold Time (min) Run Time (min)
9. Initial 80 2 2
10. Ramp1 10 180 2 14
11. Ramp2 2 220 5 39
12. Ramp3 5 240 5 48

GC-MS SETTINGS FOR PARTIALLY METHYLATED ALDITOL ACETATES:

Carrier Gas: Helium

Inlet Conditions:

• Temperature: 220 o C

• Pressure: 9.855 psi

• Flow: 22.24 mL/min

1. GC-MS Transfer Line Temp: 280 o C
2. MS Source: 230 o C
3. MS Quad: 150 o C
4. Column: DB-5 or Equivalent, 30 m x 0.25 mm x 0.25 µm
5. Column Flow: 10396 mL/min
6. Injection Volume: 1 µL
7. Run Time 49.667 min
8. Conditions o
9. C/min o
10. C Hold Time (min) Run Time (min)
11. Initial 80 0 0
12. Ramp1 5 120 1 9
13. Ramp2 3 230 4 49.667

We hope that this catalog fulfills these goals and promotes the application of discoveries to the biomedical community.

Copyright © 1996-99 by GlycoTech Corporation. All rights reserved.