Oxidation of Cysteine to Cystine

 

Important Functional Groups in this experiment

 

R-NH2             amine

 

R-CO2H          carboxylic acid

 

R-CONH2       amide

 

R-S-H             thiol

 

R-S-R             sulfide

 

R-S-S-R         disulfide

 

 

 

[Ox] = H2O2, hydrogen peroxide

 

            Proteins are natural polymers made up of amino acids linked by peptide bonds.

 

Naturally Occurring Amino Acid                             

           

 

With the exception of the amino acid glycine, where R = H, all amino acids are chiral.

 

If R is a lower priority than -CO2H and NH2, the absolute configuration is S.

 

 

Wade: Table 24-2 p.  1155-6 lists the 20 amino acids commonly found in proteins.

 

 

 

 

 

In proteins, the amino group of 1 amino acid is attached to the carboxylic acid group of another amino acid.

 

 

The amide functional group is formed.

 

peptide: dipeptide

peptide bond

 

Proteins are often referred to as polypeptides.

 

 

 

R= any of the structures found for the naturally occurring amino acids.

 

                                                                                                            disulfide bond

 

 

 

 

 

separate polypeptide chains

 

 

If the thiol groups of the cysteine units (residue) on two different polypeptide chains are oxidized, the result is the formation of a disulfide between the 2 polymer chains. 

 

The 2 polymer chains are now linked, and the material is said to be a crosslinked polymer.

 

Crosslinked polymers tend to be stronger materials than single strand polymers?  If there is a relatively small amount of crosslinking, the material is elastic (natural rubber, neoprene, etc.).  If there is a large amount of crosslinking, the material becomes tough and had strong mechanical properties.

 

 

 

 

 

Hair contains the protein keratin, which contains a relatively high percentage of cystine.

 

Decomposition

 

Some compounds decompose before they melt. Usually, at the decomposition point, the solid darkens rapidly, and bubbling is observed. Using a sealed m.p. tube, the decomposition point of cystine should be observed at 235º C.  The method for sealing m.p. tubes will be demonstrated to small groups.

 

The IR spectrum will be obtained.  One way of obtaining IR spectra of solids is to use a Nujol Mull.

 

                                                           IR Spectra of Solids

 

            1. KBr pellet

            2. Nujol Mull

            3. Solution IR

 

Nujol Mull

 

            1. Grind 5 mg. of sample into a fine powder using a mortar and pestle.

            2. Add 1-2 drops of Nujol (mineral oil).

            3. Grind it again until the mixture is a fine dispersion. (Nujol Mull) (Looks like thin milk)

            4. Place the mull between the salt plates.

            5. Obtain the IR spectrum.

            6.  Clean the plates with methylene chloride and hexanes.

 

Nujol (mineral oil): mixture of high m.w. alkanes

 

Absorb » in same regions as C-H vibrations of sample.

(See Fig. 19-10 in manual, p. 854)

The Nujol peaks obscure any peaks that might be due to C-H vibration of sample. Mark peaks due to Nujol.

 

If too much Nujol is used, then the peaks of the compound being analyzed will be hard to distinguish from even the weakest peaks Nujol.

 

Amino acids are found in 3 forms.

 

1. free amino acid (zwitterion)

 

A. 3100 - 2600 cm-1 (s, broad) NH3+ stretching vibration + other complex vibrations with one sharp band between 2222 - 2000 cm-1

B. 1660 - 1590 cm-1 (w) NH3+ asymmetric bending

1550 - 1485 cm-1 (s) NH3+ symmetric bending

C. 1650 - 1590 cm-1 (s) asymmetric stretch of carboxylate anion

1400 cm-1 (w) symmetric stretch of carboxylate anion

 

2. hydrochloride (or other salt)

 

A. 3333 - 2380 cm-1 superimposed N-H and O-H stretching vibrations

B. 1610 - 1590 cm-1 (w) NH3+ asymmetric bending vibration

1550 - 1481 cm-1 (s) NH3+ symmetric bending vibration

C. 1220 - 1190 cm-1 (s) C-O stretching in the carboxylic acid

D. 1755 - 1730 cm-1 (s) C=O stretch for amino acids

 

3. Sodium (or other cation) salts

A. 3400 - 3200 cm-1 N-H stretch normally found in amines

B. 1600 - 1590 cm-1 , 1400 cm-1 vibrations due to carboxylate anion

 

 

You will be asked to identify whether the product is in the free amino acid, hydrochloride, or metal salt form.

 

Three spectra have been posted near the IR instrument.  One is a typical background spectrum.   A spectrum of Nujol is provided.   It shows the absorptions of Nujol so that you won't confuse them for the peaks associated with your product cystine.  Finally, the third spectrum is that of cystine in Nujol.  A good spectrum of your product should look something like it. 

 

 

When you look at them, notice that the Nujol peak near 3000 cm-1 is off scale.  Normally no peak should be cut off like that in a good spectrum.  However, since the actual absorptions of cystine are all substantially weaker than the Nujol peaks in that region and since we are not concerned with Nujol, it is acceptable change the display limits so that the peaks around 1600 cm-1 appear larger.  None of those   peaks are off scale in the example.

 

They are not labeled in the example, but for the report, some of the peaks must be labeled.  This can be partially accomplished by using the Find option. For cystine, you will want to set the threshold so that all the peaks between 1300 and 1650 cm-1 will be listed.  For the posted spectrum, a good threshold value would be 40.  At that value, the small peak ~ 2100 cm-1 will not be listed and you will need to label it yourself (using the mouse and keyboard).  Also, you will also have to separately label the broad absorption ~ 2700 cm-1. 

 

Note some of the peaks may not match up perfectly.  The above values are general positions for amino acids.  In the report, you will match your peaks up as best as possible to determine the form of the amino acid present.

 

To get the best results, err on the side of above 5 mg. cystine when making the Nujol mull.

 

KBr Pellets

 

KBr does not absorb between 4000 and 600 cm-1.

 

A solid solution of the sample in KBr can be prepared by placing a physical mixture of the substances under pressure.

 

The sample will be held in a thin disk of KBr (pellet).

 

 

                                                         KBr Pellet Preparation

 

1. Dry KBr by storing in an oven. (If H2O present, broad absorption band between 3300 - 3500 cm-1.)  KBr is currently stored in the ovens in the prep room.

2. Mix 1 - 2 mg of sample with 100 mg KBr.

3. Grind the mixture (5 min.) into fine powder in an agate mortar and pestle.

4. Place portion of sample in die.

5. Tighten bolts (30-40 foot pounds on torque wrench, too much can shear bolts)

6. Leave the die under pressure for 60 sec.

7. Carefully loosen and remove bolts. A translucent disk (KBr pellet) should remain in the center of the die.

8. If the pellet is clear enough, place it in the holder.

9. Obtain IR spectrum.

 

                                                    Reasons for Cloudy Pellets

 

1. KBr mixture not ground enough

2. Sample was not dry

3. Sample: KBr ratio high

4. Pellet too thick

5. Bolts not tightened sufficiently.

6. Sample has low m.p.

 

The ends of the bolts are polished surfaces. Avoid scratching.