373A cable

Keith Rose (Keith.Rose@medecine.unige.ch)
Fri, 10 Dec 1999 16:46:59 +0100

Question from a colleague of Jan Luczo:
"We would like to modify our protein, to introduce a single aldehyde
that is (relatively) stable. Preferably we'd like to be able to do this
reproducibly at a defined location (e.g. the N- or C- terminal). The
reason for a single modification is that we do not want to run the risk of
destroying too many protective antibody epitopes on the protein by the
addition reaction. Also, the chemistry used in the addition
reaction/stabilization has to be pretty mild - we have 6 disulfide
bridges in the protein (11 kDa) that we do not want to touch. The sort of
avenue we've been exploring is to react glyceraldehyde with our protein
(addition at primary amines). If we play with the ratios/conditions we can
get to a stoichiometry of about one addition per protein molecule. We then
stabilize the resultant Schiff base to lock the molecule in place by a mild
reduction that doesn't effect the disulfides. Subsequent treatment with
sodium
periodate produces an aldehyde from the glyceraldehyde. This approach may
have limited success, but we're looking for ideas for other approaches.
For example we have thought of synthesizing a short peptide that has an
aldehyde introduced during synthesis, and then conjugating it to the
protein. However, we're not sure of the conjugation chemistry to use to
leave the aldehyde alone.
If anyone has any ideas, it would be greatly appreciated. One option of
particular appeal to me is that the protein has a C-terminal His x 6
tag. So any attack that could produce aldehydes around those Histidines would
be ideal, as it would leave the rest of the protein alone (and provide a
nice way to separate modified from unmodified!). PS I am not an organic
chemist, so nice explanations of the chemistry involved appreciated!"
___________________________________________________________________________

Suggestions from Keith Rose:
1. Aldehydes. Aldehyde chemistry is beautiful, but it a pity to use
reductive alkylation since there is always a danger of stabilizing unwanted
Schiff base links between a Lys side-chain and the aldehyde to give
cross-linked species and/or aggregates. Better is to use aldehydes to form
hydrazones or oximes. Hydrazones, once formed, can be stabilized by
reduction without much danger of cross-linking to Lys side-chains. Better
still, oximes are generally stable enough not to need stabilization through
reduction, so one does not have such problems at all.
2. Glyceraldehyde. If you reductively alkylate glyceraldehyde onto a Lys
side-chain, you get HO-CH2-CH2-NH-CH2-etc. When you treat this with
periodate you do not get a protein-bound aldehyde: the oxidation gives
formaldehyde, and (transiently) HO-CH-NH-CH2-etc, which rapidly hydrolyses
to a second mole of formaldehyde and NH2-CH2-etc, i.e. the modification is
reversible, as shown by K. Geoghegan and H. Dixon.
3. Site-specific. There are several options for introducing aldeyde
chemistry with site-specificity: (a) reverse proteolysis with e.g.
1,3-diamino-propan-2-ol followed by periodate; (b) periodate on N-terminal
Ser or Thr --> N-terminal O=CH-CO-NH-CHR-CO-etc where R is the side-chain
of residue 2; (c) alkylation with a reagent such as Ser-NHCH2CH2NH-CO-CH2I
followed by periodate. Otherwise, one can place an aminooxy group on a
protein using e.g. Br-CH2CO-NHCH2CH2NH-COCH2ONH2, and react the modified
protein with an aldehyde-containing protein (or reagent) to make a conjugate.

All this carbonyl chemistry works very smoothly, in our hands and in the
hands of others (Tam, Mutter, Kent, Offord, Geoghegan, Dawson, and others).
For some of our own work, see e.g.
46. Preparation of well-defined protein conjugates using enzyme-assisted
reverse proteolysis; Rose, K., Vilaseca, L.A., Werlen, R., Meunier, A.,
Fisch, I., Jones, R.M.L. and Offord, R.E.; Bioconjugate Chem. 2 (1991) 154-159
47. Site-specific modification of a fragment of a chimaeric monoclonal
antibody using reverse proteolysis; Fisch, I., K¸nzi, G., Rose, K. and
Offord, R.E.; Bioconj. Chem. 3 (1992) 147-153
48. Construction of protein analogues by site-specific condensation of
unprotected fragments; Gaertner, H.F., Rose, K., Cotton, R., Timms, D.,
Camble, R. and Offord, R.E.; Bioconj. Chem. 3 (1992) 262-268
54. Protein conjugates of defined structure: synthesis and use of a new
carrier molecule; Vilaseca, L.A., Rose, K., Werlen, R., Meunier, A.,
Offord, R.E., Nichols, C.L. and Scott, W.L.; Bioconj. Chem. 4 (1993) 515-520
57. Facile synthesis of homogeneous artificial proteins; Rose, K.; J. Am.
Chem. Soc. 116 (1994) 30-33
58. Chemo-enzymic backbone engineering of proteins: site-specific
incorporation of synthetic peptides that mimic the 64-74 disulfide loop of
granulocyte colony-stimulating factor; Gaertner, H.F., Offord, R.E.,
Cotton, R., Timms, D., Camble, R. and Rose, K.; J. Biol. Chem. 269 (1994)
7224-7230
60. Site-specific conjugation of an enzyme and an antibody fragment;
Werlen, R. C., Lankinen, M., Rose, K., Blakey, D., Shuttleworth, H.,
Melton, R. and Offord, R.E.; Bioconj. Chem. 5 (1994) 411-417
62. High-yield, site-specific coupling of N-terminally modified
beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab';
Mikolajczyk, S.D., Meyer, D.L., Starling, J.J., Law, K., Rose, K. and
Offord, R.E.; Bioconj. Chem. 5 (1994) 636-646
67. Site-specific immunoconjugates; Werlen, R.C., Lankinen, M., Smith, A.,
Chernushevich, I., Standing, K.G., Blakey, D.C., Shuttleworth, H., Melton,
R.G., Offord, R.E. and Rose, K.; Tumor Targeting 1 (1995) 251-258
68. In vitro and in vivo comparison of a randomly coupled antibody
fragment-enzyme conjugate with a site-specific conjugate; Werlen, R.C.,
Offord, R.E., Blakey, D.C., East, S.J., Melton, R.G. and Rose, K.; Biomed.
Peptides Prots. Nucl. Acids 1 (1995) 251-254
70. Preparation of a trivalent antigen-binding construct using polyoxime
chemistry: improved biodistribution and potential for therapeutic
application; Werlen, R.C., Lankinen, M., Offord, R.E., Schubiger, P.A.,
Smith, A. and Rose, K.; Cancer Res. 56 (1996) 809-815
71. Natural peptides as building blocks for the synthesis of large
protein-like molecules with hydrazone and oxime links; Rose, K., Zeng, W.,
Regamey, P.-O., Chernushevich, I., Standing, K.G. and Gaertner, H.F.;
Bioconj. Chem. 7 (1996) 552-556
78. Plasmodium falciparum polyoximes: highly immunogenic synthetic vaccines
constructed by chemoselective ligation of repeat B cell epitopes and a
universal T cell epitope of CS protein; Nardin, E.H., Calvo-Calle, J.M.,
Oliveira, G.A., Clavijo, P., Nussenzweig, R., Simon, R., Zeng, W. and Rose, K.
Vaccine 16 (1998) 590-600
81. Stepwise solid-phase synthesis of polyamides as linkers; Rose, K. and
Vizzavona, J.; J. Am. Chem. Soc. 121 (1999) 7034-7038
82. Totally synthetic lipid containing polyoxime peptide constructs are
potent immunogens; Zeng, W., Jackson, D.C., Murray, J., Rose, K. and
Brown, L.E.; Vaccine 18 (1999) 1031-1039
83. New cylcization reaction at the Amino terminus of peptides and
proteins; Keith Rose et al., Bioconj. Chem. 10 (1999) 1038-1043

3. Polypeptide and protein derivatives and a process for their preparation
Offord, R.E. and Rose, K.
European Patent EP 024 3929 B1 (27 Sep. 1995) and corresponding
Australian, Japanese and other Patents
4. Polypeptide derivatives and a process for their preparation
Offord, R.E. and Rose, K.
European Patent EP 0359 428 B1 (9 Aug. 1995)
7. Cluster conjugates of drugs with antibodies
Koppel, G.A., Offord, R.E., Rose, K. and Scott, W.L.
US Patent 5,272,253, Dec. 21 1993
8. Homogeneous Multivalent Molecules, Methods of Using and Making Same
Rose, K. and Offord, R.E.
US Patent Application 08/057,594 filed May 5, 1993; notice of allowance
received
9. Homogeneous Polyoxime Compositions and Their Preparation by Parallel
Assembly
Rose, K. and Offord, R.E.
US Patent Application 08/114,877 filed August 31, 1993; notice of
allowance received
10. Hetero-Polyoxime Compositions and Their Preparation by Parallel Assembly
Rose, K. and Offord, R.E.
US Patent Application 08/105,904 filed August 31, 1993; notice of
allowance received
11. Polyoxime compounds and their preparation
Rose, K. and Offord, R.E.
PCT Patent Application filed May 4th, 1994
12. Compositions for insulin-receptor mediated nucleic acid delivery
Thatcher, D.R., Offord, R.E., Rose, K. and Gaertner, H.F.
US Patent 5,830,852. Nov. 3 1998

I hope that is helpful,
Keith ROSE, PhD