Nucleotide analogs and process for making same enzyme

Patent metadata

Field	Value
Publication number	US-9534243-B2
Application number	US-201414174981-A
Country	US
Kind code	B2
Filing date	Feb 7, 2014
Priority date	Feb 7, 2013
Publication date	Jan 3, 2017
Grant date	Jan 3, 2017

Abstract

A process for making a nucleotide analog includes combining a first substrate that includes a linker and a base with a second substrate to form a substrate composition. An enzyme contacts the substrate composition and catalyzes formation of the nucleotide analog from the first substrate and the second substrate. Additionally, a composition includes the first substrate, second substrate, the enzyme, the nucleotide analog, and optional additives.

First claim

What is claimed is:

1. A process for making a nucleotide analog, the process comprising:
combining a first substrate comprising a compound of formula 1
P 3 —P 2 —P 1 -L-Q  Formula 1

with a second substrate comprising a compound of formula 27
R-Q 1 -R 5   Formula 27

to form a substrate composition;
contacting the substrate composition with an enzyme; and
catalyzing, with the enzyme, formation of the nucleotide analog of formula 28 from the first substrate and the second substrate
R-Q 1 -P 1 -L-Q  Formula 28

wherein the enzyme comprises an amino acid sequence having at least 90% identity to the amino acid sequence consisting of

(SEQ ID NO. 1)

MSKLLREVTPEERRLYYSGEWDAKKLPEFIVESIERREFGFDHTGEGPSD

RKNAFSDVRDLEDYIRATAPYAAYSSVAFYRNPQEMEGWLGAELVFDIDA

KDLPLRRCQNEHPSGQVCPICLEDAKELARDTLIILKEDFGFENIHVVYS

GRGYHIRVIDEWALKLDSKARERILSYVSAAEEVTFDDIQKRYIMLSSGY

FRVFRLRFGYFIQRINENHLKNIGLKRSTAEKLLDEKTRQDIVEKFVNKG

LLAAFPEGVGYRTLLRLFGLSTTFSKAYFDGRVTVDLKRILRLPSTLHSK

VGLVATYIGSDEKRLEKFDPFKDAVPEFRKEEVQKAYQEWKELHEG;

L is a linker comprising a structure of formula 3, formula 6, formula 7, formula 8, formula 9, formula 10, formula 11, or formula 12,

wherein * is a point of attachment;
Q 2 and Q 3 are independently O, S, Se, NR, CR 2 , or C═CR 2 ;
Q 4 is O, S, NR, CR 2 , CR 2 CR 2 , CR 2 O, CR 2 OCR 2 , CR 2 S, CR 2 SCR 2 , CR 2 NR, CR 2 NRCR 2 , alkenylene, alkylene, alkyleneoxy, alkynylene, amide, aralkylene, arylene, aryleneoxy, cycloalkylene, fluoroalkylene, heteroaralkylene, heteroarylene, heterocycloalkylene, or a single bond;
Q 5 is N or CR;
R 1 , R 2 , R 2′ , and R 4 are independently R, OR, SR, NR 2 , NROR, NRNR 2 , N 3 , NO 2 , CHO, CN, C(═O)NH 2 , or C(═O)OR; alternatively, R 2 and R 2′ together are ═O, ═S, ═N—R, or ═CR 2 ; and
R is independently H, F, Cl, Br, I, OH, SH, NHOH, NHNH 2 , CHO, C(═O)OH, alkenyl, alkenyleneamine, alkoxy, alkyl, alkyleneamine, alkynyl, amine, amino, aralkyl, aralkyloxy, aralkyloxy, aryl, aryleneamine, aryloxy, carbocyclic, carboxylic acid group or salt, cycloalkyl, cycloalkyloxy, haloalkyl, heteroaralkyl, heteroaryl, or heterocycloalkyl;

Q is a base comprising a structure of formula 13, formula 14, formula 15, or formula 16,

wherein A 1 , A 2 , A 3 , A 4 , A 5 , and A 6 are independently N, C—R 1 ;
A 7 , A 8 , A 9 is independently N—R 1 , C(R 1 ) 2 , C═O, C═C—(R 1 ) 2 , C═N—R 1 ; and
R 1 is as defined above;

P 1 and P 2 are respectively a first phosphate group and a second phosphate group independently having a structure of formula 25, and P 3 is a third phosphate group having a structure of formula 26,

wherein Q 6 is O, NR, or S; and Q 4 , R, and * are as defined above;

Q 1 is O, S, Se, NR, CR 2 , or C═CR 2 , cycloalkenylene, cycloalkylene, heterocycloalkenylene, heterocycloalkylene; and
R 5 is H, F, Cl, Br, I, OH, SH, NHOH, NHNH 2 , CHO, alkenyleneamine, alkyleneamine, amine, aryleneamine, or carboxylic acid group or salt.

2. The method of claim 1 , wherein the base Q comprises

3. The method of claim 2 , wherein the base Q comprises

4. The method of claim 1 wherein the linker L comprises

5. The method of claim 4 , wherein the linker L is

6. The method of claim 4 , wherein P1 and P2 independently have a structure of formula 31, and P3 has a structure of formula 32

7. The method of claim 1 , wherein the second substrate is an alcohol, an amino acid, a chromophore, a fatty acid, a sugar, or a combination comprising at least one of the foregoing.

8. The method of claim 1 , wherein the second substrate is 2-(N-morpholine) ethanesulfonic acid (MES), N-(2-acetamido) iminodiacetic acid (ADA), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), (2-aminoethyl)-trimethyl ammonium chloride hydrochloride (Cholamine), N,N-bis(2-hydroxy-ethyl)-2-aminoethane sulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), tris(hydroxymethyl) aminomethane (TRIS), N-tris(hydroxyl-methyl)methylglycine (Tricine), N,N-bis(2-hydroxyethyl)-glycine (Bicine), 2-(N-cyclohexylamino) ethane-sulfonic acid (CHES), acetic acid, phosphoric acid, citric acid, ethylenediaminetetraacetic acid, a salt thereof, or a combination comprising at least one of the foregoing.

9. The method of claim 1 , wherein the nucleotide analog comprises

10. The method of claim 9 , wherein the nucleotide analog is

wherein R 8 is a functional group selected from H, CH 3 ,

11. The method of claim 1 , wherein the nucleotide analog is Tris-dAMP having the structure

or glycerol-dAMP having the structure

12. The method of claim 1 , wherein the enzyme is a single subunit or a complex comprising a small subunit and a large subunit arranged in a complex.

13. The method of claim 1 , wherein the substrate composition further comprises an alkaline earth metal cation, a transition metal cation, or a combination comprising at least one of the foregoing in an amount effective so that the enzyme has activity with respect to catalyzing formation of the nucleotide analog.

14. The method of claim 1 , further comprising immobilizing the first substrate, the second substrate, the enzyme, or a combination comprising at least one of the foregoing on a solid phase support during

Assignees

• Marino John P
• Kelman Zvi
• Hurwitz Jerard
• Giulian Gary G
• Us Commerce
• Memorial Sloan Kettering Cancer Center
• Univ Maryland

Inventors

• Marino John P
• Kelman Zvi
• Hurwitz Jerard
• Giulian Gary G

Classifications

• C12P19/32 Primary

  having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12P19/305

  Pyrimidine nucleotides · CPC title

• C07H19/207

  the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title