CA1244786A - Synthesis of amino-derivitized oligonucleotides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention consists of a method for the synthesis of oligonucleotides which contain free aliphatic amino group(s).
The synthetic method is general, permitting amino groups to be placed on oligonucleotides of any composition or length which is attainable by current DNA synthetic methods.

Description

7~6 sACKGROuND OF THE INVENTION
An oligonucleotide is a short polymer consisting of a linear sequence of four nucleotides in a defined order. The nucleotide subunits are joined by phosphodiester linkages joining the 3' hydroxyl moiety of one nucleotide to the 5' hydroxyl moiety of the next nucleotide. An example of an oligonucleotide is 5' ApCpGpTpApTpGpGpCp 3'. The letters A, C, G and T refer to the nature of the purine or pyrimidine base coupled at the l-position of deoxyribose. A, adenine; C, cytosine; G, guanine; T, thymidine.
P represents the phosphodiester bond.
The single stranded oligonucleotides of this invention are further characterized by being homogenous with respect to the ; sequence of the nucleoside subunits and are of uniform molecular weight.
Synthetic oligonucleotides are powerful tools in modern molecular biology and recombinant DNA work. There are numerous applications for these molecules, including a) as probes for the isolation of specific genes based on the protein sequence of the gene product, b) to direct the in vitro mutagenesis of a desired gene, c) as primers for DNA synthesis on a single-stranded template, d) as steps in the total synthesis of genes, and many more, reviewed in Wm. R. Bahl et al, Prog. Nucl. Acid Res. Mol. Biol., 21, 101 (1~78).
; A very considerable amount of effort has therefore been

-2~

--~ ~ 7~36 1 devoted to the development of efficient chemical methods for 2 the synthesis of such oligonucleotides. A brief review of

3 these methods as they have developed to the present is

4 found in Crockett, G. C., Aldrichimica Acta 16(3), 47-55 (1983).

The best methodology currently available utilizes the phosphor-6 amidite derivatives of the nucleosides in combination with a 7 solid phase synthetic procedure, Matteucci et al, J. Am. Chem.
Soc., 103, 3185 (1981); and Beaucage et al, M. ~. Tet. Lett., 9 22(20), 1858-1862 tl981). Oligonucleotides of length up to 30 bases~may be made on a routine basis in this matter, and 11 molecules as long as 50 bases have been made. Machines that 12 employ this technology are now commercially available.
13 There are other reports in the literature of the derivi-14 tization of DNA, A modified nucleoside triphosphate has been developed wherein a biotin group is conjugated to an aliphatic 16 amino group at the 5 position of uracil, Langer et al, Proc.
17 Nat. Acad. Sci., U.S.A., 78, 6633-6637 (1981). This nucleotide 18 derivative is effectively incorporated into double stranded DNA.
19 Once in DNA it may be bound by anti-biotin antibody which can then be used for detection by fluorescence or enzymatic methods.
21 The DNA which has had biotin conjugated nucleosides incorporated 22 therein by the method of Langer et al is fragmented into smaller 23 single and double stranded pieces which are heterogeneous with 24 respect to the sequence of nucleoside subunits and variable in 25 molecular weight. Draper and Gold~ Biochemistry, 19, 1774-1781 26 (1980), reported the introduction of aliphatic amino groups by a 27 bisulfite catalyzed transamination reaction, and their subsequent 28 reaction with a fluorescent tag. In Draper and Gold the amino ., ~;~gL47~

1 group is attached directly to a pyrimidine base. The amino group 2 so positioned inhibits hydrogen bonding and for this reason, these 3 materials are not useful in hybridization and the like. Chu et al, 4 Nucleic Acid Res. 11~18), 6513-6529 (1983), have reported a method for attaching an amine to the terminal 5'phosphate of oligonucleo-tides or nucleic acids.
7 There are many reasons to want a method for covalently attaching other chemical species to synthetic oligonucleotides.
9 Fluorescent dyes attached to the oligonucleotides permits one to elimi~ate radioisotopes from the research, diagnostic and 11 clinical procedures in which they are used, and improve shelf-12 life and availability. As described in the assignee's co-13 pending application for a DNA sequencing machine (Serial No.
14 the synthesis of fluorescent-labeled oligonucleotides permits the automation of the DNA sequer.cing process. The development 16 of appropriate techniques and instrumentation for the detection 17 and use of fluorescent-labeled oligonucleotides allows the auto-18 mation of other currently laborious laboratory and clinical 19 techniques. The attachment of DNA cleavage chemicals such as those disclosed by Schult-z et al, J. Am. Chem. Soc., 104, 6861 21 (1982); and Hertzberg et al, J. Am. Chem. Soc., 104, 313 (1982) 22 permits the construction of synthetic restriction enzymes, 23 whose specificity is directed by the oligonucleotide sequence.
2L~ This present invention presents a general method for the introduction OL a free aliphatic amino group(s) into 26 synthetic oligonucleotides. This amino group is readily and 27 specifically reacted with a variety of amino reactive function-28 alities, and thereby permits the covalent attachment of a wide 47~6 variety of chemical species.
In the attached figures, Figure 1 represe.nts a typical structure or a section ~: of an oligonucleotide, Figure 2 represents the molecule of 5'-trifluoro-acetamido 5'-deoxy 31-N,N-diisopropyl phosphoramido thymine, and Figure 3 represents a reaction sequence to prepare this compound, discussed in Examples I and II.

-5-47~6 6829g-77 SUMMARY OF THE INVENTION
Briefly, the present invention comprises novel aliphatic aminoderivitized single stranded oligonucleotides conjugated to a detectable moiety which is a chromophore, fluorescent agent, proteln, enzyme or other "tag".
This invention further includes the novel oligo-nucleotides having inserted therein at least one aminoderivitized nucleo~ide via phosphoramidite precursor.
In another aspect, this invention comprehends the synthesis of oligonucleotides on a solid phase ~upport, wherein the oligonucleotide is reacted with a protectedamino-derivitized nucleoside phosphoramidite.
; In accordance with the present invention, there is provided novel protected 5'-amino nucleoside phosphoramidites having the generic formula:

o CF - C - N - CH

H H ~ H
O H

R2o N(Rl)2 wherein B is a nucleoside base or nucleoside base analog and Rl and R2 are lower alkyl.
The present invention also provides an improved method of the synthesis of 5'-amino oligonucleotides which comprises ~'~

47~36 682~9-77 reacting a phosphoramidite compound having the general formula:
~: O
CF3 - C - 1- C~ ~ B

H

R20 N(Rl)2 wherein B is a nucleoside base, or nucleoside base analog, and Rl and R2 are lower alkyl in the last coupling step in solid phase oligonucleotide synthesis with an oligonu-cleotide bound to a solid support.
The invention is inDnespecific aspect, the synthesized molecule 5'-trifluoroacetamido 5'-deoxy 3'-N,~-diisopropyl phosphoramido thymidine (Figure 2) and its addition to the 5' terminus as the last addition in solid phase oligo-nucleotide synthesis. During cleavage and deprotection of the oligonucleotidel the trifluoroacetyl group is hydrolyzed, leaving a free aliphatic amino group on the 5' terminus of the ; 20 oligonucleotide. This amino-derivitized oligonucleotide may then be reacted with any of a wide variety of amino-reactive ~` molecules to give the corresponding oligonucleotide derivative.
- This is a special case of the more general approach of using a ::
modified nucleoside which has a protected aliphatic amino group on the base moiety, rather than on the 5' carbon. Such a molecule allows several free amino groups to be placed within the oligonucleotide, and at any desired position in the oligonucleotide - 6a -.

~ ~ 47~36 1 sequence.
It is an object of this invention to provide new reagents 3 and techniques applicable to DNA sequencing.
4 It is also an object of the invention to provide improve-ments in DNA hybridization for the detection of~genetic diseases

6 and for other purposes.

7 These and other objects and advantages of my invention

8 will be apparen~ to those skilled in this art from the more

9 ¦ specif~c d sclosure which follows.

2sl ~,., ~ 7~36 3 The strategy used to introduce aliphatic amino groups 4 into an oligonucleotide is to synthesize a 3'phosphoramidite derivative of a nucleoside analogue containing a protected 6 aliphatic amino group. This phosphoramidite may then be reacted 7 with the oligonucleotide being synthesized on a solid support in a manner analogous to the reaction of underivitized nucleoside 9 phosphoramidites Cleavage from the solid phase and deprotection of the base moieties and aliphatic amino group yields the 11 amino-derivitized oligonucleotide.

Synthesis of VI. In Figure 3, the synthesis of compound 16 VI from the commercially available compound I (thymidine~ is 17 shown. The synthesis of compounds II-IV are disclosed in Horo-18 wit et al, J. Org. Chem., 27, 3045-3048 (1962); and Gibbs and ~rge , 19 J. Carbohydrates-Nucleosides and Nucleotides, 3(5 and 6), 315-334 (19761. The synthesis of compounds V and VI are as follows.

24 5'trifluoroacetamido 5'deoxythymidine(V): 1.25 gr _ .
(5 mmoles) of 5'-amino 5'deoxythymidine was dissolved in 25 ml 26 dry dimethyl formamide. To this was added 1.3 ml (10 mmoles) 27 S-ethyl trifluorothioacetate (Aldrich). The reaction was stirred 28 gently at room temperature. TLC of the reaction mixture on . .

~ 1 ~49~7~36 1 ¦ silica gel F-254 plates run in MeOH:Acetone 1:1 show a single ¦ spot of product detected by short wave UV. The product has 3 ¦ a high mobility in this solvent system in contrast to the start-4 ¦ ing compound VI which is virtually immobile 5 ¦ The reaction mixture was rotary evaporated to dryness ¦ under reduced pressure, transferred to an Erlenmeyer flask in 7 30 ml isopropanol, and recrystallized from boiling Isopropanol:
8 MeOH. Yield = 1.315 gr ~3.9 mmoles, 80% yield), m.p. 261-262 9 (dec), anal. pred. C, 42.7~; H, 4.18~ N,12.5%, exp. C, 42.7~, H, 4.16%; N~ 12.4%. The structure of V was ~urther confirmed by 11 'H NMR.

This Example illustrates the preparation of a protected 16 aminoderivitized nucleoside phosphoramidite.
17 5'trifluoroacetamido 5'-deoxy 3'-N,N-diisopropyl phos-18 phoramido thymidine (VI): All glassware, syringes and capillary 19 tubes used in this reaction were baked overnight in a drying oven.
Dimethyl formamide (DMF) was stored over 4 A molecular sieves 21 (Linde). Diisopropyl ethyl amine (DIPEA) was distilled from potas-22 sium hydroxide, then from calcium hydride, an~ stored over 4 ~ molecular sieves 23 To a dry 3-necked 50 ml round-bottom flask with stlr bar 24 was added 63 mg (0.19 mmoles) V. The three necks were plugged with rubber serum stoppers and needles inserted in the stoppers.
26 The flask was pumped on for several hours in a dessicator over 27 dry CaC12. The flask was kept under a gentle stream of dry 28 nitrogen gas and 2 ml of dry DMF added by syringe. 60 ~1 of _g_ ,, ~ 47~36 1 DIPEA (0.34 mmoles) was added in a dry 100 ~l capillary tube.
2 40 111 of chloro-N,N-diisopropyl amino methoxy phosphine 3 (American Bionuclear, Emeryville, CA) was added (in a dry 100 4 ~l capillary tube). The reaction was stirred gently until all starting material was dissolved, and then allowed to sit at 6 room temperature (always under N2). TLC after one hour on 7 silica gel F-254 plates in HCCL3:EtOH:Et3N 88:10:2 showed a single spot of product of much higher mobility than the 9 starting material V. Attempts to purify this product in a fashion similar to that described for the nucleoside phosphoramidites (~) 11 were unsuccessful due to degradation of the product. Therefore, 12 the crude reaction mixture was used directly for the coupling 13 to the synthetic oligonucleotide on the solid phase support.
14 The structure of VI is inferred from the reactivity of this product in the addition to the oligonucleotide, and from the 16 expected product of the reaction based on literature results.

18 EX~MPLE IV

; This Example illustrates the preparation of an oligonucleo-21 tide coupled at the 5' terminus via a phosphodiester linkage to 22 the 3' hydroxyl of 5'-amino 5'-deoxythymidine.
23 Addition of VI to the 5'terminus of an oligonucleotide-24 A base-protected synthetic oligonucleotide of sequence 5'OH-AGC
ACT TTT AGA GT 3' coupled to the solid phase at the 3' terminus 26 was prepared by methods which are described in detail in the 27 protocol entitled "A Procedure for the Manual Synthesis of 28 Deoxyoligonucleotides using dimethoxytrityl nucleoside phos-

-10-L7~36 phoramidites on a Solid Support", issued by Applied Biosystems, 2 Inc.l Foster City, California. The differences for reaction of 3 the oligonucleotide with VI are a) in place of steps 4.22 4 and 4.23, 1 ml of the freshly prepared reaction mixture contain-ing VI is combined with 1 ml of 0.5M tetrazole in acetonitrile 6 and added to the reaction vessel; b) after step 4.33 there are 7 two 30-second washes with acetonitrile; and c) the capping 8 section 5 is omitted (so as to allow for assay of unreacted 9 hydroxyl groups in a subsequent addition).
The efficiency of the coupling reaction is readily monitore

11 by subsequent coupling with a "normal" phosphoramidite and cleavage

12 of the dimethoxy trityl group to give a color assay. If the 5'

13 hydroxyl groups of the oligonucleotide reacted in the first coup-ling with VI, they are no longer available or reaction in the subsequent coupling, and there will be little color released upon 16 DCA treatment following the second coupling. In this example 17 OD450=1.12 (after dilution) for the DMT group released from the 18 oligonucleotide prior to reaction with VI. OD450=0.026 (after di-19 lution) for the DMT released from a G residue added subseauent to reaction with VI. Therefore 98% of the 5' OH groups were blocked 21 by reaction with VI. The oligonucleotide was deprotected and 22 cleaved `from the solid phase by treatment with thiophenol 23 and concentrated NH40H in the usual manner, and the NH40H
24 removed under reduced pressure. The oligonuclectide was dissolved in l ml of distilled H20. The OD260 was 128 of this solution, 26 indicating the concentration of DNA to be 4.5 mg/ml. 50~1 27 of this solution was diluted to l ml with H20 and mixed for 28 15 min with a few hundred mg of AG50~-X4(sodium form) ion ,~.

~2~7~36 1 exchange resin to convert the DNA from the ammonium salt to 2 the sodium salt (the ammonium ions interfere with ~he subse-3 quent ninhydrin assay). The resin was removed by centrifugation, 4 and the supernatant dried down in a Savant rotary concentrator.
Quantitative ninhydrin assay(Sarin, V. K., et al, Anal. Biochem.
6 117, 147-157 (1981)), gave approximately 1 mole of amino group 7 per mole oligonucleotide (the molar concentration of oligonucleo-8 tide was determined from a calculated extinction coefficient 9 E260 = 1.55 x 105, based on nucleotide composition). Ninhydrin assay on~the same molar amount of a control oligonucleotide to 11 which no VI had been conjugated gave no amino positive-reaction.

14 Conjugation with dye: To 100 ~1 of the above solution 16 of amino oligonucleotide was added 200 ~1 H2O, 50 ~1 of 1 M
17 carbonate/bicarbonate buffer pH 9.0, and 25 ~1 of freshly prepared 18 10 mg/ml fluorescein isothiocyanate (FITC)(Molecular Probes, Inc., 19 Junction City, Oregon) in dimethyl sulphoxide. The mixture was left at room temperature for several hours, and purified by 21 chromatography on a column (1 cm x 9 cm) of Sephadex G-25 22 medium in H2O. The yellow product eluted in the excluded vol-23 ume and was cleanly resolved from unreacted dye. A control 24 reaction with oligonucleotide to which no VI had been reacted gave very little or no color in the excluded volume of the 26 column, indicating that the dye was indeed reacting with the 27 added amino group. The dye-oligonucleotide conjugate had 28 D260 = 2-3, OD4gs = 0-54. Based on E495 7 x 104 for FITC, 7~6 1 this gives a 7.7 ~M solution of dye. Based on E260 = 1.55 x 2 105, the DNA is 12.8 ~M. Therefore ~60% of the DNA molecules were labeled with dye. This is a rough estimate, which does 4 not allow for changes in the bound fluorescein absorption relative to unbound, nor for contaminating shorter and non-6 reactive oligonucleotides. An aliquot of this colored DNA was 7 electrophoresed on a 20% polyacrylamide gel and was clearly 8 visible as a single colored (and fluorescent) band of a mobility 9 appropriate for an oligonucleotide of that length.
Th~e dye conjugated oligonucleotide was readily purified 11 by high performance liquid chromatography (HPLC) on a reverse 12 phase Clg column (Waters) using an acetonitrile:0.1 M triethyl 13 ammonium acetate pH 7.0 gradient for elution.
14 There are numerous possible applications of the novel amino-derivitized oligonucleotides which have been disclosed above. The 16 aliphatic amino group is easily and specifically reacted with a 17 large number of functional groups. This means that virtually any 18 desired molecule may be attached to an oligonucleotide prepared 19 as described above. This includes enzymes, other proteins, fIuorescent tags, bioluminescent tags, chromophores, and so on.
21 Oligonucleotides have been widely used in a number of areas, often 22 in conjunction with radiolabels. It will be possible to substitute 23 non-radioactive probe molecules for the radioactive labels. This 24 will make procedures utilizing oligonucleotides cheaper and easier to use, and compatible with a clinical setting. While 26 radiolabels are less preferred, the novel amino-derivitized 27 oligonucleotides can also be radiolabeled, for example, such as 28 with I125. The followingthree particular examples of uses of the ~ `
~ -13-j ~t -` I ~LZ~47~6 1 ¦ novel amino-derivitized oligonucleotides are illustrative only:
2 1 l. Automated DNA sequencing.
3 ¦ 2. Detection of genetic disease by DNA hybridization.
4 ¦ 3. General use of fluorescence for detection of hybridiza-5 ¦ tion.
6 ¦ Detection of Genetic Abnormalities: Oligonucleotides 7 ¦ ma~ be used to determine the genotype of individuals ~his is 8 ¦ done on DNA samples obtained from the fetus by aminiocentesis.
¦ This information is invaluable for the ~enetic counseling of couples at risk for a variety of ~enetic diseases. The genotype ll of adults may also be determined, allowing effective diagnosis 12 and treatment at an early stage. One striking example of this 13 technology is for the detection of sickle cell anemia, Connor et 14 al., Proc Natl. Acad. Sci. USA 80, 278 (1983). Mineteen-base-pair-long synthetic oligonucleotides were synthesized, one com-16 plementary to the normal human ~-globin gene (~A), and one com-17 plementary to the sickle cell ~-globin gene (~S). These molecules 18 were radioactively labeled and used as probes in D~A hybridization.
19 Under appropriate hybridization conditions, these probes can be used to distinguish the ~A gene from the ~ allele. This allows 21 diagnosis of the sickle cell disease. More generally, as pointed 22 out in Connor et al, "this allele-specific h~bridiæation behavior 23 of oligonucleotides provides a general method for the diagnosis of 24 any genetic disease which involves a point mutation in the DNA
sequence of a single-copy gene." The present invention is directly 26 applicable to this technique. The oligonucleotide probes are pre-27 pared with an amino group and labeled with a fluorescent tag. The 28 fluorescent probe molecule is stable indefinitelv, in contrast to :.
-l4-LZ~4786 1 the short lifetime of radioactive probes an~ requires no special 2 precautions for use or handling. This makes such an approach 3 vastly preferable for use in a clinical setting, which is the 4 major area in which the technology will be used.
Oligonucleotide probes are widely used in research 6 work as well as clinical work. They are commonly used to detect 7 a piece of DNA and a desired sequence in a "libraryj" a collection 8 of DNA fragments cloned into a plasmid or phage vector, which 9 contains sequences encompassing the entire genome (or expressed RNA) of an organism. They are also used to hybridize to DNA of a 11 given sequence in a "blot" of a restriction digest of a particu-12 lar piece of DNA. In all these examples, as well as others, the 13 oligonucleotide is labeled with 32p, usually at the 5'terminus, 14 and the molecules are detected by autoradiography. The present invention describes the labeling of oligonucleotides with fluor-16 escent dyes. Therefore, fluorescence may be used for detection o~
17 the molecules in any of the techniques in which radioactivity has 18 been conventionally used. This present numerous advantages over 19 radioactivity such as stability of the probes, expense and ease of use and disposal.
21 Having fully described the invention it is intended that 22 1 it be limi only by the lawful scope of the appended claims.

Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Novel protected 5'-amino nucleoside phosphor-amidites having the generic formula:

wherein B is a nucleoside base or nucleoside base ana-log and R1 and R2 are lower alkyl.

2. An improved method of the synthesis of 5'-amino oligonucleotides which comprises reacting a phosphoramidite compound having the general formula:

wherein B is a nucleoside base, or nucleoside base ana-log, and R1 and R2 are lower alkyl in the last coupling step in solid phase oligonucleotide synthesis with an oligonu-cleotide bound to a solid support.

3. The product of Claim 2.

4. The method of Claim 2 wherein the oligonucleotide is subsequently cleaved from the support.

5. The product produced by the method of Claim 4.

6. The product of Claim 5 conjugated to a detectable moiety.

7. The product of Claim 6 wherein the detectable moiety is a fluorophore.

8. The product of Claim 6 wherein the detectable moiety is a chromophore.

9. The product of Claim 6 wherein the detectable moiety is a protein.

10. The product of Claim 6 wherein the detectable moiety is an enzyme.

11. The product of Claim 6 wherein the detectable moiety contains radioactive I125.

12. The compound 5' -trifluoroacetamido-5' -deoxy-3'N, N-diisopropyl phosphoramido thymidine.

13. In the preparation of oligonucleotides containing an aliphatic amino group at the 5' terminus, the improvement wherein the aliphatic amino group is introduced by reacting an amino nucleoside phosphoramidite in the last coupling step of oligonucleotide synthesis on a solid phase support.

14. In the preparation of oligonucleotides containing an aliphatic amino group at the 5' terminus, the improvement wherein the aliphatic amino group is introduced by reacting a protected 5' -amino-5' -deoxy-3' phosphoramido thymidine in the last coupling step of oligonucleotide synthesis on a solid phase support.

15. The preparation of oligonucleotides containing an aliphatic amino group at the 5' terminus, the improvement wherein the aliphatic amino group is introduced by reacting a 5' -tri-fluoroacetamido-5'-deoxy-3' -phosphoramido thymidine in the last coupling step of oligonucleotide synthesis of a solid phase support.