The Development of a Plasma Volume Expander 67 



Our two, and only, previous trials with conjugate had been conducted 

 with dogs as test animals. Although the material injected had been 

 an unfractionated solution of sodium salt of conjugate containing some 

 unreacted peptide, two conclusions were drawn from the experiments: 

 (a) the conjugate remained longer in the blood stream than did the 

 straight peptide and (6) the conjugate we had made was not large 

 enough to give the desired duration of blood-stream retention. The 

 number average molecular weight of backbone in these conjugates 'had 

 been 12,000-13,000 and that of the side chain had been smaller. The 

 side chains had been obtained by methylation of the peptide by sus- 

 pending the pure dry peptide of molecular weight 12,000 in dry 

 methanolic HC1 for 24 hours, removing methanol and HG1 in vacuo, 

 and precipitating the ester from methanol solution with ether. During 

 this procedure the peptide was degraded, the extent of degradation 

 varying with the precise conditions of reaction. 



Methods were therefore also developed for preparing side chains from 

 the peptide with no degradation in chain length. By working under 

 controlled conditions of time, temperature, and moisture, it was found 

 possible to prepare a suitable polymethyl ester of peptide by adding 

 ethereal diazomethane to an ethereal suspension of peptide. If meth- 

 ylation is allowed to proceed to completion, there is generally methyl- 

 ation of practically all of the terminal amino groups and loss of their 

 availability for conjugation. Too little methylation gives a pyridine- 

 insoluble product. The best compromise seemed to be a product 

 methylated to the extent of 50-60%. This is usually 60-70%> soluble 

 in anhydrous pyridine and retains 80-90% of the end groups as un- 

 methylated amino groups. There is no degradation of the peptide in 

 this process. 9 



The backbone polyhydrazide was prepared as previously by full 

 methylation of another sample of peptide. This is easily achieved by 

 methylation with diazomethane as above, except that a small amount 

 of methanol is added to the reaction, and the methylation is allowed 

 t<> go to completion, as evidenced by absence of free carboxyl on titra- 

 tion of a small filtered aliquot, with dilute alkali in the presence of 

 phenolphthalein indicator. 



Excess diazomethane is discharged by addition of ethereal formic 

 acid, and the ester is filtered off and dried. On dissolving in 50%) 

 methanolic hydrazine the ester is converted to polyhydrazide which 

 is precipitated out by addition of methanol. This in turn is converted 

 into polyazide by conventional methods at — 10°C. Polyazide, which 

 is a white water-insoluble powder at this temperature, is soluble and 



