Hydrochloric Acid → 1-Cyano-4-(dimethylamino)pyridin-1-ium tetrafluoroborate

HRID 2364939Notes

Reaction equation

HRID 2364939 reaction equation
[F:1][B-:2]([F:5])([F:4])[F:3].C([C:8]1C=CC=[CH:10][N+:9]=1N(C)C)#N.[N:17]#[C:18]Br.[Na+].[Cl-].Cl.[OH-].[Na+].NCC(O)=O.OC1O[C@H:38]([CH2:40]O)[C@@H:36](O)[C@H:34](O)[C@@H:32]1[NH2:33].OC1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1N.O=C[C@@H]([C@H]([C@@H]([C@@H](CO)O)O)O)O.O=C[C@H]([C@H]([C@@H]([C@@H](CO)O)O)O)O>C1N(CCO)CCN(CCS(O)(=O)=O)C1.CS(C)=O.C(N(CC)CC)C>[B-:2]([F:5])([F:4])([F:3])[F:1].[CH3:8][N:9]([C:36]1[CH:34]=[CH:32][N+:33]([C:18]#[N:17])=[CH:40][CH:38]=1)[CH3:10]

Participants

14

Reactant 10

HCID 313Hydrochloric AcidHCID 750Glycine2×HCID 5234Sodium Chloride2×HCID 10476Cyanogen BromideHCID 14798Sodium HydroxideHCID 107526(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanalHCID 161658DemannoseHCID 439213D-GlucosamineHCID 440049D-MannosamineHCID 87094273HCID 87094273, compound3×

Product 1

HCID 98811511-Cyano-4-(dimethylamino)pyridin-1-ium tetrafluoroborate

Reagents, catalysts & solvents 3

HCID 679Dimethyl SulfoxideSolventHCID 8471TriethylamineSolventHCID 238314-(2-Hydroxyethyl)-1-piperazineethanesulfonic acidSolvent3×

Procedure

This example describes a generalized method for making insulin-glycogen conjugates using cyanodimethylamino-pyridinium tetrafluoroborate (CDAP) as a coupling agent. This synthesis was used to prepare various insulin-glycogen conjugates that are described in later Examples. The inventors have found that this method produces increased insulin loading as compared to the CNBr method of Example 2. Briefly, 8.0 g of glycogen is dissolved in 160 ml of 25 mM HEPES, 0.15 M NaCl, pH 9.0. 2.4-12.0 mL of a 1.0M CDAP solution in DMSO is added dropwise to the glycogen solution at 0° C. After stirring for 1 minute, a volume equal to that of the CDAP solution consisting of 0.2M triethylamine (TEA) is added dropwise over one minute. At this time, the pH of the reaction solution is adjusted to 9.0 using 1.2N HCl. Then, 80-2000 ml of a 10 mg/ml insulin solution in a 20 mM HEPES (pH 9.0) solution is added over the next three minutes and the pH adjusted again to 9.0 using 0.3N NaOH. Finally, in some cases 1.6 ml of a 100 mg/ml glycine solution in 100 mM HEPES buffer containing 0.15M NaCl (pH 9.0) can be added. In other cases, amine-functionalized sugars such as mannosamine, glucosamine, and other glucose or mannose-containing sugars. The inventors have found that the addition of the glycine step increases the solubility of the conjugate. The entire reaction mixture is slowly stirred at room temperature overnight.

Workup

  1. customproduces
  2. temperatureincreased insulin loading
  3. additionis added dropwise over one minute
  4. additioncan be added
  5. temperatureincreases the solubility of the conjugate
  6. customThe entire reaction mixture
  7. stirringis slowly stirred at room temperature overnight

Sources & evidence

ORD record
ord-ef64bc7a48184b4b8051196098da7621
Source dataset
uspto-grants-2014_04
Patent number
US08697643B2

Reaction notes

Loading…