IP Library › Granted Patent US 12,540,162
Granted Patent B2
US 12,540,162 · App. 18/235,184 · Granted Feb 3, 2026

Preparation of functional homocysteine residues in polypeptides and peptides

Inventors: Timothy J. Deming (Los Angeles, CA); Eric G. Gharakhanian (Los Angeles, CA)
Assignee: The Regents of the University of California
C07K7/06C07K1/006C07K1/107C07K1/113C07K1/1133C07K14/001C08G69/10C08G69/48
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Quick Facts
Patent No.
US 12,540,162
App. No.
18/235,184
Granted
Feb 3, 2026
Kind
B2
Abstract

Methodology was developed for transformation of methionine residues into homocysteine derivatives. Methionine residues can undergo alkylation reactions at low pH to yield sulfonium ions, which can then be selectively demethylated to give alkyl homocysteine residues. This process tolerates many functional groups.

Claims (44)

1 . A method of preparing a polypeptide comprising one or more RC H residues, wherein each R—C H residue in the polypeptide has a structure represented by formula (I):

wherein:

R X , independently for each —C H residue, is H or alkyl; and R, independently for each R—C H residue, is substituted or unsubstituted alkyl, provided that R is not unsubstituted methyl, allyl, benzyl, oligoethylene glycol, or glycosylated alkyl, and further provided that R does not contain sulfoxide; comprising contacting an M R sulfonium residue represented by formula (II) with a resonance stabilized anionic nucleophile, in the presence of an organic solvent that is miscible with water, thereby demethylating the M R sulfonium residue and forming the polypeptide comprising one or more R—C H residues, wherein each R—C H residue in the polypeptide has a structure represented by formula (I) and wherein M R has a structure represented by formula (II):

and

further wherein R is less electrophilic and more sterically demanding relative to the methyl group of formula II.

2 . The method of claim 1 , wherein the nucleophile is ammonium pyrrolidinedithiocarbamate (APDC).

3 . The method of claim 1 , wherein the nucleophile is thioacetate.

4 . The method of claim 1 , wherein contacting an M R sulfonium residue with a nucleophile further comprises contacting the M R sulfonium residue with ethanol.

5 . The method of claim 1 , wherein demethylating the M R sulfonium residue favors demethylation of the M R sulfonium residue by at least 75%, as compared to dealkylation of the M R sulfonium residue.

6 . The method of claim 1 , wherein demethylating the M R sulfonium residue favors demethylation of the M R sulfonium residue by at least 90%, as compared to dealkylation of the M R sulfonium residue.

7 . The method of claim 1 , wherein the polypeptide is a homopolymer.

8 . The method of claim 1 , wherein the polypeptide is a heteropolymer.

9 . The method of claim 1 , wherein the polypeptide comprises at least 4 residues.

10 . The method of claim 1 , wherein the polypeptide is

11 . The method of claim 1 , wherein R is substituted or unsubstituted propyl.

12 . The method of claim 1 , wherein R has a structure represented by:

wherein R′ is selected from alkoxy, azido, aryl, heteroaryl, halo, allyloxy, alkylcarbonyl, phosphonate, carbamate, amido, NH 3 + ,

13 . The method of claim 1 , wherein the R—C H residue has a structure represented by:

wherein:

R 2 is selected from H, alkyl, aminoalkyl, acyl, and alkoxy-C(O)—; and

n is an integer from 0-10.

14 . The method of claim 13 , wherein:

R x is H;

R 2 is H, C 1-3 alkyl, C 1-3 aminoalkyl, or Ac; and

n is 1, 2, or 3.

15 . The method of claim 1 , wherein R is substituted or unsubstituted 2-hydroxypropyl.

16 . The method of claim 1 , wherein R has a structure represented by:

wherein R′ is selected from alkoxy, azido, aryl, heteroaryl, halo, allyloxy, alkylcarbonyl, phosphonate, carbamate, amido, NH 3 + ,

17 . The method of claim 1 , wherein the R—C H residue has a structure represented by:

wherein:

R 1 is selected from H, alkyl, acyl, and alkoxy-C(O)—;

R 2 is selected from H, alkyl, acyl, and alkoxy-C(O)—; and

n is an integer from 0-10.

18 . The method of claim 17 , wherein:

R x is H;

R 1 is H;

R 2 is H, C 1-3 alkyl, or Ac; and

n is 1, 2, or 3.

19 . The method of claim 1 , wherein the polypeptide comprises

and

wherein X + is a counterion.

20 . The method of claim 1 , wherein R is substituted or unsubstituted butyl.

21 . The method of claim 1 , wherein R is substituted or unsubstituted ethyl.

22 . The method of claim 1 , wherein the polypeptide comprises at least 10 residues.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 11, 2023
From: DEMING, TIMOTHY J.; GHARAKHANIAN, ERIC G.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 065828/0754 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 8, 2023
From: DEMING, TIMOTHY J.; GHARAKHANIAN, ERIC G.
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 065519/0237 →
Continuity (3)
Continuation 16096951
Provisional Application 62328394 · Apr 27, 2016
Related Publication 20240092833A1 · Mar 21, 2024
References Cited (71)
US 4165332A · Beard et al. · 1979 [cited by applicant]
US 5599903A · Kauvar et al. · 1997 [cited by applicant]
US 7132475B2 · Hubbell et al. · 2006 [cited by applicant]
US 9718921B2 · Deming et al. · 2017 [cited by applicant]
US 10351591B2 · Deming et al. · 2019 [cited by applicant]
US 10526396B2 · Lecommandoux et al. · 2020 [cited by applicant]
US 11732008B2 · Deming et al. · 2023 [cited by applicant]
US 20100222407A1 · Segura et al. · 2010 [cited by applicant]
US 20110177508A1 · Bestor et al. · 2011 [cited by applicant]
US 20110223217A1 · Dixon et al. · 2011 [cited by applicant]
US 20140294932A1 · Kim et al. · 2014 [cited by applicant]
US 20190119322A1 · Deming et al. · 2019 [cited by applicant]
CN 104926924A · 2015 [cited by applicant]
EP 0226827A2 · 1987 [cited by applicant]
EP 0761203A1 · 1997 [cited by applicant]
EP 1712557A1 · 2006 [cited by applicant]
JP 2004231633A · 2004 [cited by applicant]
WO WO1996040757A2 · 1996 [cited by applicant]
WO WO2009151708A2 · 2009 [cited by applicant]
WO WO2010023670A2 · 2010 [cited by applicant]
WO WO2013082116A1 · 2013 [cited by applicant]
WO WO2013148727A1 · 2013 [cited by applicant]
WO WO2016154120A1 · 2016 [cited by applicant]
WO WO2017021334A1 · 2017 [cited by applicant]
WO WO2017189860A1 · 2017 [cited by applicant]
WO WO2022261181 · 2022 [cited by applicant]
U.S. Appl. No. 14/770,417, Granted. [cited by applicant]
U.S. Appl. No. 15/559,981, Granted. [cited by applicant]
U.S. Appl. No. 15/748,806, Granted. [cited by applicant]
Alferiev et al., “High reactivity of alkyl sulfides towards epoxides under conditions of collagen fixation—a convenient approach to 2-amino-4-butyrolactones,” Biomaterials, 22(18):2501-2506 (2001). [cited by applicant]
Brown et al., “Strategy for “Detoxification” of a Cancer-Derived Histone Mutant Based on Mapping Its Interaction with the Methyltransferase PRC2,” Journal of The American Chemical Society, 136(39):13498-13501 (2014). [cited by applicant]
Catalog page for 2 bromoethyl triflate from ABX, http://web.archive.org/web/20090706013707/http://abx.de/chemicals/6182.html, available online Jul. 2009. [cited by applicant]
Chilkoti et al., “Targeted drug delivery by thermally responsive polymers,” Advanced Drug Delivery Reviews, 54(5): 613-630 (2002). [cited by applicant]
Extended European Search Report for EP Application No. EP 17790439 dated Nov. 13, 2019. [cited by applicant]
Extended European Search Report issued by the European Patent Office, dated Jan. 28, 2016, in related Application No. EP 15306247. [cited by applicant]
Gharakhanian et al., “Chemoselective synthesis of functional homocysteine residues in polypeptides and peptides,” Chem Commun, 52(30): 5336-5339 (2016). [cited by applicant]
Gharakhanian et al., “Role of side-chain molecular features in tuning lower critical solution temperatures (LCSTs) of oligoethylene glycol modified polypeptides,” J Phys Chem B, 120(26): 6096-6101 (2016). [cited by applicant]
Gharakhanian et al., “Versatile Synthesis of Stable, Functional Polypeptides via Reaction with Epoxides,” Biomacromolecules, 16(6):1802-1806 (2015). [cited by applicant]
Hanson et al., “Nonionic block copolypeptide micelles containing a hydrophobic rac-leucine core,” Macromolecules, 43(15):6268-6269 (2010). [cited by applicant]
Hayakawa, T. et al., “Syntheses and conformational studies of poly (S-aminoalkyl-homocysteine)s and their benzyloxycarbonyl derivatives”, Polymer Journal, 7(5): pp. 538-543 (1975). [cited by applicant]
Huang et al., “Biologically active polymersomes from amphiphilic glycopeptides,” J Am Chem Soc, 134:119-22 (2011). [cited by applicant]
International Search Report and Written Opinion for International Application No. PCT/EP2016/068232 dated Nov. 14, 2016. [cited by applicant]
International Search Report and Written Opinion for International Application No. PCT/US2013/033938 dated Jul. 22, 2013. [cited by applicant]
International Search Report and Written Opinion for International Application No. PCT/US2017/029867 dated Jul. 20, 2017. [cited by applicant]
International Search Report and Written Opinion for International Application No. PCT/US22/032637 dated Oct. 12, 2022. [cited by applicant]
International Search Report from corresponding International Application No. PCT/US2014/018763, mailed Jun. 2, 2014. [cited by applicant]
International Search Report from corresponding International Application No. PCT/US2016/023428, mailed Jun. 29, 2016. [cited by applicant]
Jamonnak et al., “Substrate specificity of SIRT1-catalyzed lysine N?-deacetylation reaction probed with the side chain modified N?-acetyl-lysine analogs,” Bioorganic Chemistry, 38(1):17-25 (2010). [cited by applicant]
Kaplowitz, et al., “The importance and regulation of hepatic glutathione,” Yale J Biol Med, 54: 497-502 (1981). [cited by applicant]
Kramer et al., “Glycopolypeptide conformations in bioactive block copolymer assemblies influence their nanoscale morphology,” Soft Matter, 9(12):3389-95 (2013). [cited by applicant]
Kramer et al., “Reversible chemoselective tagging and functionalization of methionine containing peptides,” Chemical Communications, 49:5144-5146 (2013). [cited by applicant]
Kramer, “Multimodal Switching of Conformation and Solubility in Homocysteine Derived Polypeptides,” J. Am. Chem. Soc, 136: 5547-5550, (2014). [cited by applicant]
Kramer, Jessica R., et al. “Preparation of Multifunctional and Multireactive Polypeptides via Methionine Alkylation,” Biomacromolecules, 13: 1719-23 (2012). [cited by applicant]
Kultyshev, et al., “S-Alkylation and S-Amination of Methyl Thioethers—Derivative of closo-[B12H12]2-. Synthesis of a Boronated Phosphonate, gem-Bisposphonates, and Dodecaborane-ortho-carborane Oligomers,” J Am Chem Soc,… [cited by applicant]
Kyte, et al., “Purification of peptides that contain methionine residues,” Method Enzymol, 91: 367-377 (1983). [cited by applicant]
March, Jerry Advanced Organic Chemistry (1992) ISBN 0-471-60180-2, p. 294-298 and p. 352-354. [cited by applicant]
Notice of Allowance and Fees Due for U.S. Appl. No. 15/559,981 dated Feb. 25, 2019. [cited by applicant]
Pande, et al., “Suppression of phase separation in solutions of bovine lambda IV-crestallin by polar modification of the sulfur-containing amino acids,” PNAS, 88(11): 4916-4920 (1991). [cited by applicant]
Reid, et al., “Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry,” J Am Soc Mass Spectr, 16(7): 1131-1150 (2005). [cited by applicant]
Ribeiro et al., “Influence of the amino-acid sequence on the inverse temperature transition of elastin-like polymers,” Biophys J, 97:312-20 (2009). [cited by applicant]
Roemmele et al., “Chirospecific synthesis of beta-hydroxy alpha-amino acids,” The Journal Of Organic Chemistry, 54(8): 1866-1875 (1989). [cited by applicant]
Ross et al., “A straightforward preparation of primary alkyl triflates and their utility in the synthesis of derivatives of ethidium,” Journal of the Chemical Society Perkins Transactions 1, 1(4): 571-574 (2000). [cited by applicant]
Stark, et al., “Alkylation of the methionine residues of ribonuclease in 8M urea,” J Biol Chem, 269(11): 3755-3761 (1964). [cited by applicant]
Storer, et al., “Aracyl triflates for preparing fluorescent and UV absorbing derivatives of unreactive carboxylates, amines, and other reactive metabolites,” Analytica Chimic Acta, 558: 319-325 (2006). [cited by applicant]
Supplementary European Search Report dated Sep. 16, 2016 from EP 14 75 7627. [cited by applicant]
Taichi, et al., “Suppression of side reactions during final deprotection employing a strong acid in boc chemistry: regeneration of methionyl residues from their sulfonium salts,” Int J Peptide Res Ther, 15(4): 247-253 (… [cited by applicant]
Teeuwen et al., “‘Clickable’ elastins: elastin-like polypeptides functionalized with azide or alkyne groups,” Chem Comm, 4022-4 (2009). [cited by applicant]
Toennies, et al., “Methionine Studies VII. Sulfonium Derivatives,” Journal of the American Chemical Society, vol. 67, 1945, pp. 849-851. [cited by applicant]
Umemura, et al., “Alylation of several nucleophiles with alkylsulfonium salts,” Bull Chem Soc Japan, 63: 2593-2600 (1990). [cited by applicant]
Urry et al., “Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity,” Journal of the American Chemical Society, 113(11): 4346-4348 (1991). [cited by applicant]
Weeden et al., “A retro-inverso α-melanocyte stimulating hormone analog with MC1R-binding selectivity,” Journal of Peptide Science, 17:47-55 (2011). [cited by applicant]