Method of using colored phosphorylating reagents

A method of phosphorylating a nucleoside or an oligonucleotide chain having a free 2', 3' or 5' hydroxyl moiety is provided. The method involves the use of a phosphorylating reagent which is selected such that the extent of phosphorylation can be monitored colorimetrically, easily and accurately. The phosphorylating reagent contains an aromatic species such as a dimethoxytrityl group that is cleavable with acid and colorimetrically detectable upon release. Examples of phosphorylating reagents which are useful in conjunction with the disclosed method include ##STR1##

TECHNICAL FIELD 
The invention relates to chemical phosphorylation reagents and more 
particularly relates to chemical phosphorylation reagents useful in DNA 
synthesis and purification. 
BACKGROUND 
With the advent of hybrid DNA technology and the explosion in the ability 
to isolate, purify, and assay a wide variety of natural products, both 
polypeptides and nucleic acids, there is an increasing need for rapid and 
efficient methods of preparing and purifying oligomers of amino acids and 
nucleic acids. 
With nucleic acids, it is typically necessary to synthesize sequences for 
use as linkers, adapters, synthetic genes and synthetic regulatory 
sequences, as well as for use as probes, primers, and the like. Many 
procedures have been developed for producing oligomers of nucleotides. 
These procedures for the most part rely on initial attachment of a first 
nucleotide to a solid support by a selectively cleavable linkage, followed 
by sequential addition of subsequent nucleotide units, with each addition 
involving a number of chemical reactions. 
The two primary methods of oligonucleotide synthesis which are 
well-established in the art are the so-called "phosphotriester" and 
"/phosphoramidite" methods (described at some length in the references 
cited below). In the most prevalent schemes for both methods, the 
oligonucleotide chain grows by nucleophilic attack of the 5'-OH of the 
immobilized oligomer on an activated 3,-phosphate or phosphoramidite 
function of a soluble 5,-protected nucleotide building block. Other key 
steps include the acid deprotection of the 5,-O-(4,4,dimethoxytrityl) 
group (DMTr) in the phosphotriester method, and, in the phosphoramidite 
process, the oxidation of the phosphite triester to the phosphate 
triester. 
Other methods of oligonucleotide synthesis are also known, including 5' to 
3' syntheses which use a cyanoethyl phosphate protecting group (De Napoli 
et al., Gazz. Chim. Ital. 114:65 (1984); Rosenthal et al., Tetrahedron 
Lett. 24:1691 (1983); Belagaje and Brush, Nucleic Acids Res. 10:6295 
(1977)) and solution phase 5' to 3' syntheses (e.g., Hayatsu and Khorana, 
J. Amer. Chem. Soc. 89:3880 (1967); Gait and Sheppard, Nucleic Acids Res. 
4:1135 (1977); Cramer and Koster, Angew. Chem. Int. Ed. Engl. 7:473 
(1968); and Blackburn et al., J. Chem. Soc. C, 2438 (1967)) 
After completion of oligonucleotide synthesis and deprotection of the 
product, the free 5'-OH group of the oligonucleotide must be 
phosphorylated or phosphitylated for use in most biological processes. 
Also, phosphorylation or phosphitylation on the 3'-OH function is 
typically necessary to generate oligonucleotides in a form that can be 
purified, stored and/or commercialized. See Sonveaux, Bioorganic Chem. 
14:274,294 (1986) The present invention is directed to compounds which are 
useful in phosphorylating and phosphitylating both 3' and 5' hydroxyl 
moieties. 
5' -phosphorylation is generally carried out with T4 polynucleotide kinase 
and ATP, a reaction that is not particularly reliable or efficient. 
Several methods for chemical 5'-phosphorylation are also known, including 
that described in Nadeau et al., Biochemistry 23:6153-6159 (1984), van der 
Marel et al., Tetrahedron Lett. 22:1463-1466 (1981), Himmelsbach and 
Pfleiderer, Tetrahedron Lett. 23:4793-4796 (1982), Marugg et al., Nucleic 
Acids Res. 12:8639-8651 (1984), and Kondo et al., Nucleic Acids Research 
Symposium Series 16:161-164 (1985). However, most of these methods involve 
the use of unstable reagents or require extensive modification of standard 
deprotection and purification procedures. Similar problems have been found 
with monofunctional and bifunctional 3'-phosphorylating reagents (see 
Sonveaux, supra, at 297). 
The present invention is directed to novel phosphorylating reagents which 
overcome the limitations of current phosphorylation procedures. As used 
herein, the term "phosphorylating reagent" encompasses compounds which can 
phosphorylate a hydroxyl group directly as well as phosphitylating agents 
which, when coupled with a subsequent oxidation step, can phosphorylate 
hydroxyl groups indirectly, i.e., in a two-step reaction sequence. The 
phosphorylating reagents disclosed herein are also useful in the method of 
the parent application hereto, U.S. Ser. No. 891,789 (now abandoned), 
which is directed to a method of synthesizing and purifying 
oligonucleotides substantially free of erroneous sequences. The disclosure 
of that application is hereby expressly incorporated by reference in its 
entirety. 
The reagents of the present invention, especially the phosphitylating 
reagents as will be described, are advantageous in that they are easily 
accommodated by currently available DNA synthesis machines. Also, the 
phosphorus blocking groups designated herein as Y, Y' or Y', are easily 
removed during deprotection of the completed oligonucleotide and do not 
require any additional deprotection steps. Most importantly, the reagents 
disclosed herein provide for rapid and accurate online monitoring of 
oligonucleotide synthesis. That is, the present compounds yield a leaving 
group upon deprotection of the completed oligonucleotide which is readily 
observable. 
DESCRIPTION OF THE PRIOR ART 
In addition to the art cited above, Matteucci and Caruthers, J. Am. Chem. 
Soc. 103:3185-3191 (1981), describe the use of phosphorchloridites in the 
preparation of oligonucleotides. Beaucage and Caruthers, Tetrahedron Lett. 
22:1859-1862 (1981) and U.S. Pat. No. 4,415,732 describe the use of 
phosphoramidites in the preparation of oligonucleotides. Smith, ABL 15-24 
(Dec. 1983), describes automated solid phase oligodeoxyribonucleotide 
synthesis. See also the references cited therein, and Warner et al., DNA 
3:401-411 (1984), whose disclosure is incorporated herein by reference. 
Fisher and Caruthers, Nucl. Acids Res. 11(5): 1589-1599 (1983), describe a 
procedure for monitoring the progress of a deoxynucleotide synthesis. That 
procedure involves monitoring the release of various triarylmethyl groups 
during synthesis, each of which is "color coded," i.e., are differently 
colored in acid solution. 
Amidine protection of adenosine has been described by McBride and 
Caruthers, Tetrahedron Lett. 24:245 (1983) and Froehler and Matteucci, 
Nucl. Acids Res. 11:8031 (1983) Other blocking groups are described in 
co-pending application Ser. No. 891,789, the parent application hereto. 
Horn and Urdea, DNA 5(5):421-425 (1986) describe phosphorylation of 
solid-supported DNA fragments using 
bis(cyanoethoxy)-N,N-diisopropyl-aminophosphine. 
DISCLOSURE OF THE INVENTION 
In one aspect of the invention, the invention encompasses phosphitylating 
reagents having the structure 
##STR2## 
wherein: R.sub.1 may be virtually any group whose release upon 
phosphorylation and nucleotide deprotection can be monitored, e.g., 
colorimetrically. R.sub.1 is preferably a compound having the formula 
RR'R"C-- wherein the R, R' and R" are independently selected from the 
group consisting of 
##STR3## 
wherein the X.sub.1 and X.sub.2 may be ortho, meta or para to each other 
and are typically hydrogen, lower alkyl, lower alkoxy, halogen, nitro, 
phenyl, sulfonate, or amines substituted with from 0 to 2 lower alkyl or 
lower alkoxy substituents. X.sub.1 and X.sub.2 may also be part of a 
polycyclic aromatic system having typically from one to five rings, such 
as phenyl, naphthyl or the like. In the latter case, X.sub.1 and X.sub.2 
carbon atoms which are para to each other in the higher conjugated 
aromatic structure, and the rings may be unsubstituted or substituted with 
one or more of the aforementioned substituents. R.sub.2 is selected from 
the group consisting of methylene optionally mono- or disubstituted with 
lower alkyl and phenyl optionally substituted with lower alkyl or nitro. Y 
is selected from the group consisting of amino substituted with from 0 to 
2 lower alkyl groups, halogen trialkylsilyl of from 3 to 12 carbon atoms, 
and heterocyclic moieties typically having a total of from 1 to 3, usually 
1-2, heteroannular members and from 1 to 3 rings, and x is an integer in 
the range of 1 and 50 inclusive. D is selected from the group consisting 
of (i) the structure II: 
##STR4## 
in which J.sub.1, and J.sub.2 are independently selected from the group 
consisting of hydrogen and alkyl of 1 to 3 carbon atoms, c is 0 or 1, and 
Q is typically selected from the group consisting of hydrogen, alkyl of 
from 1 to 9 carbon atoms, nitro, alkylsulfonyl (generally lower 
alkylsulfonyl), arylsulfonyl, cyano, p-nitrophenyl, alkylthio (generally 
lower alkylthio), arylthio, trihalomethyl, and (ii)phenyl, beta-naphthyl, 
9-fluorenyl and 2-anthraquinonyl. 
The invention also encompasses phosphitylating reagents given by the 
structure III and phosphorylating reagents given by the structure IV: 
##STR5## 
wherein R.sub.1, R.sub.2, D and x are as given above for the reagents of 
Formula I. 
The reagents of the invention can be used either to phosphitylate 
hydroxyl-containing compounds to give phosphite triesters (i.e., using the 
reagents of Formulae I and III) or to phosphorylate hydroxyl-containing 
compounds to give phosphate triesters (i.e., using the reagent of Formula 
IV). 
Where a phosphoramidite according to formula I is used as a phosphitylating 
agent, an activating agent is typically necessary as well. Suitable 
activating agents are described, for example, in Froehler and Matteucci, 
Tetrahedron Lett., 24:3171 (1983) and Beaucage and aruthers (1981), supra. 
Where phosphitylation is effected using the reagent of Formula III, or 
where phosphorylation is carried out using the reagent of Formula IV, a 
condensing agent as described in the literature (e.g., Sonveaux, supra, or 
Froehler and Matteucci, Tetrahedron Lett. (1983), supra), is to be used. 
As noted in U.S. Pat. application Ser. No. 07/891,789 (now abandoned), the 
parent application hereto, preferred condensing agents are activated aryl 
sulfonic acid compounds such as mesitylene 
sulfonyl-3-nitro-1,2,3-triazole, or mesitylenesulfonyl chloride and 
N-methylimidazole. 
In the case of the phosphitylating reagents, it is generally desirable to 
oxidize the resulting phosphite triesters to give the corresponding 
phosphate triesters and phosphate salts. 
In another aspect, then, the invention encompasses a method of 
phosphorylating the 5'-hydroxyl group of nucleosides and oligonucleotide 
chains via the aforementioned methods. Such a method is useful subsequent 
to synthesis of an oligonucleotide chain, so that the completed sequence 
will be 5'-phosphorylated for further use. The reagents are also useful in 
providing hydroxyl blocking groups--i.e. phosphite or phosphate 
triesters--during DNA synthesis. 
Modes of Carrying Out the Invention 
1. Definitions 
By "oligonucleotide" is meant a nucleotide chain having from about 2 to 
about 100 component nucleotide monomers. 
The terms "phosphorylating conditions" or "phosphitylating conditions" as 
used herein are intended to mean reaction conditions suitable for 
substantially complete phosphorylation or phosphitylation, respectively, 
of a hydroxyl-containing compound as will be described. 
By "phosphorylating reagents" as used herein are meant compounds which 
include a phosphorous atom in the +5 oxidation state and, upon reaction 
with hydroxyl-containing compounds, yield a phosphate triester. 
By "phosphitylating reagents" as used herein are meant compounds which 
include a phosphorous atom in the +3 oxidation state and, upon reaction 
with hydroxyl-containing compounds, yield a phosphite triester. As 
phosphitylation coupled with a subsequent oxidation step is equivalent to 
a two-step phosphorylation, phosphitylating reagents may sometimes be 
referred to herein as "phosphorylating reagents." 
"Lower alkyl" and "lower alkoxy" designate alkyl and alkoxy groups, 
respectively, having from 1 to 6 carbon atoms. 
2. Structure of the Novel Reagents 
The reagents of the present invention are the phosphorylating agents 
defined by Formulae I-IV above. 
In general, the R.sub.1, R.sub.2, D, and Y substituents are as given above, 
and x is typically in the range of 1 and 50 inclusive. In a preferred 
embodiment: 
(1) The integer "x" is in the range of 1 and 8 inclusive. 
(2) R.sub.1 is 4,4'-dimethoxytrityl. 
(3) Y is an amine substituent of the formula --NT.sup.1 T.sup.2, where 
T.sup.1 and T.sup.2 may be the same or different and may be hydrocarbon or 
have from 0 to 5, usually 0 to 4 heteroatoms, primarily oxygen as oxy, 
sulfur as thio, or nitrogen as amino, particularly tert-amino, NO.sub.2, 
or cyano. The two T's may be taken together to form a mono- or 
polyheterocyclic ring having a total of from 1 to 3, usually 1 to 2 
heteroannular members and from 1 to 3 rings. Usually, the two T's will 
have a total of from 2 to 20, more usually 2 to 16 carbon atoms, where the 
T's may be aliphatic (including alicyclic), particularly saturated 
aliphatic, monovalent, or, when taken together, divalent radicals defining 
substituted or unsubstituted heterocyclic rings. The amines defined by Y 
include a wide variety of saturated secondary amines such as 
dimethylamine, diethylamine, diisopropylamine, dibutylamine, 
methylpropylamine, methylhexylamine, methylcyclopropylamine, 
ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, 
butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidine, piperidine, 
2,6-dimethylpiperidine, piperazine and similar saturated monocyclic 
nitrogen heterocycles (U.S. Pat. No. 4,415,732). 
Specific groups reported for use as --NT.sup.1 T.sup.2 are as follows: 
______________________________________ 
N-pyrrolidino Beaucage, Tetrahedron Lett. 
25:375 (1984), Schwarz and 
Pfleiderer, ibid 25:5513 
(1984) 
N = .chi..sup.1 
.chi..sup.1 - alkylene of 4-12 carbon 
atoms, -p-bis-dimethylene- 
cyclohexane, bis-diethylene 
sulfide and methylamino 
N .chi..sup.1 ; T.sup.1,T.sup.2 -Me, iPr 
McBride and Caruthers, ibid 
24:245 (1983) 
.chi..sup.1 - bis-diethyleneoxy, 
.alpha.,.alpha.,.alpha.',.alpha.'-tetramethylpenta- 
methylene 
nitroimidazole, tetrazole 
Matteucci and Caruthers, J. 
Am. Chem. Soc. 103:3185 
(1981) 
______________________________________ 
Illustrative groups include: N-pyrrolidino, N-piperidino, 
1-methyl-N-piperazino, N-hexahydroazipino, N-octahydroazonino, 
N-azacyclotridecano, N-3-azabicyclo-(3.2.2.)nonano, thiomorpholino, 
N,N-diethylamino, N,N-dimethylamino, N,N-diisopropylamino, piperidino, 
2,2,6,6-tetramethyl-N-piperidino. 
Y may also be halo, e.g., chloro (Letsinger and Lunsford, J. Am. Chem. Soc. 
(1976) 98:3655; Matteucci and Caruthers, supra) or an ammonium oxy salt, 
particularly trialkylammonium of from 3 to 12 carbon atoms. 
(4) For the most part as noted above, D is illustrated by Formula II 
wherein J.sub.1, J.sub.2 and J.sub.3 are independently selected from the 
group consisting of hydrogen and alkyl of 1 to 3 carbon atoms, c is 0 or 
1, and Q is typically selected from the group consisting of hydrogen, 
alkyl of from 1 to 9 carbon atoms, nitro, generally lower alkylsulfonyl, 
alkylsulfonyl, arylsulfonyl, cyano, p-nitrophenyl, alkylthio, generally 
lower alkylthio, arylthio, trihalomethyl, and (ii)phenyl, beta-naphthyl, 
9-fluorenyl and 2-anthraquinonyl. 
Specific groups reported for use as D are as 
______________________________________ 
alkyl Beaucage and Caruthers, 
Tetrahedron Lett. 22:1859 
(1981) 
NCCH.sub.2 C(Me).sub.0-2 (H.sub.2-0)-- 
Koster, Nucleic Acids Res. 
12:4539 (1984); Marugg et 
al., Rec. trav. Chim. Pay- 
Bays 103:97-8 (1984); Van 
Boom et al., Nucleic Acids 
Res. 12:8639 (1984) 
-p-.phi..sub.2 NOCH.sub.2 CH.sub.2 -- 
Schwarz and Pfleiderer, 
Tetrahedron Lett. 25:5513 
(1984) 
MeSO.sub.2 CH.sub.2 CH.sub.2 -- 
Claesen et al., ibid 25:1307 
(1984) 
(halo).sub.3 CC(Me).sub.0-2 (H).sub.0-2 -- 
Takaku et al., Chemistry 
Letters 1984:1267; Letsinger 
et al., Tetrahedron 40:137 
(1984) 
.phi.(CH.sub.2).sub.0-1 S(0).sub.0-2 (CH.sub.2).sub.2 
Balgobin et al., Tetrahedron 
Lett. 22:1915 (1981); 
Agarwal et al., J. Am. Chem. 
Soc. 98:1065 (1976); Felder 
et al., Tetrahedron Lett. 
25:3967 (1984) 
(.chi.).sub.0-2 OCH.sub.2 --,2-naphthyl-CH.sub.2 --, 
Caruthers et al., Nucleic 
9-fluorenyl-CH.sub.2 --, 
Acids Res. Sym. Ser. 7:215; 
2-anthraquinonyl-CH.sub.2 -- 
(1980); Christodonlon & 
Reese, Tetrahedron Lett. 
24:1951 (1983); Kwiatkowski 
et al., Abstract, Conf. on 
Syn. Oligonucleotides in 
Molecular Biology, Uppsala, 
Sweden Conf. 16-20 #64 
(1982); Balgobin, ibid 
.chi.CH.sub.2 CH.sub.2 -- 
Uhlmann et al., Tetrahedron 
Lett. 21:1181 (1980); Schulz 
and Pfleiderer, ibid 24:3582 
(1983); Beite and 
Pfleiderer, ibid 25:1975 
(1984) 
MeCOCH(Me)-- Ramirez et al., Tetrahedron 
39:2157 (1983) 
.phi..sub.3 CO(Cl) 
Vasseur et al., Tetrahedron 
Lett. 24:2573 (1983) 
______________________________________ 
X may be hydrogen or any non-interfering stable substituent, neutral or 
polar, electron donating or withdrawing, generally being of 1 to 10, 
usually 1 to 6 atoms and generally of from 0 to 7 carbon atoms, and may be 
an aliphatic, alicyclic, aromatic or heterocyclic group, generally 
aliphatically saturated, halohydrocarbon, e.g., trifluoromethyl, halo, 
thioether, oxyether, ester, amide, nitro, cyano, sulfone, amino, azo, etc. 
3. Synthesis of the Phosphorylating Reagents 
The phosphitylating reagent given by Formula I may be synthesized by a 
method analogous to that described in Horn and Urdea, Tetrahedron Lett. 
27(39):4705-4708 (1986), the disclosure of which is hereby incorporated by 
reference. 
##STR6## 
is initially provided, wherein: (1) in a first, preferred embodiment, R' 
is halogen, preferably chlorine, R" is Y as defined above, and R"' is --OD 
as defined above; (2) in a second embodiment, R' and R" are both Y and may 
or may not be identical, and R"' is --OD; and (3) in a third, alternative, 
embodiment, R', R", and R"', are all halogen, preferably chlorine. This 
compound is reacted with an alcohol of Formula VI. 
##STR7## 
under an inert atmosphere and at a relatively low temperature, preferably 
about 0.degree. C., to give a structure in which either a halogen 
substituent (embodiment (1)) or a "Y" substituent (embodiments (2) and 
(3)) has been replaced by 
##STR8## 
to give the phosphitylating agent of Formula I. (In the case of 
alternative embodiment (3), the resulting compound is caused to couple to 
a substituent "OD", followed by reaction with an amine moiety "Y" 
according to synthetic methods known in the art, to yield the structure of 
Formula I.) 
The phosphitylating reagent given by Formula III is prepared by 
condensation of the alcohol of Formula V with phosphorous acid or an 
alkylated analog thereof, in the presence of an activating agent such as 
to syl chloride. 
The phosphorylating reagent of Formula IV is prepared in a manner analogous 
to that described above for the reagent of Formula I, except that the 
starting material is 
##STR9## 
wherein: (1) in a first, preferred embodiment, R' is halogen, preferably 
chlorine, R" is --OD as defined above, and R"40 is --OH; (2) in a second 
embodiment, R' and R" are both halogen, preferably chlorine, and R"' is 
--OD; and (3) in a third, alternative embodiment, R', R"' and are all 
halogen, preferably chlorine. In embodiments (2) and (3), an additional, 
hydrolysis, step is required, while in embodiment (3) specifically, still 
a further step is required to add the "OD" substituent using synthetic 
methods known in the art. 
Isolation of the product is done via precipitation as the barium or 
triethylamine salt. 
All of these reactions are preferably carried out neat in order to 
facilitate homogeneity and to avoid problems with solubility. If desired, 
however, any suitable inert solvent may be used, providing that all 
reagents involved are substantially soluble therein. 
4. Phosphitylation and Phosphorylation Using the Novel Reagents: 
In general, the reagents disclosed herein are useful in converting free 
hydroxyl groups to phosphite and phosphate triesters. In a preferred 
embodiment, the free hydroxyl group so converted is the 5'-OH of a 
nucleoside or the 5'-OH of an oligonucleotide chain. The reaction proceeds 
according to Scheme I: 
##STR10## 
In Scheme I, the substituent "M" is a purine or pyrimidine base, which may 
be protected with amine protecting groups as disclosed in the parent 
application hereto. "E" is hydrogen, a suitable 3'-OH protecting group, or 
a continuing oligonucleotide chain. 
Typically, the reaction conditions for the phosphorylation reaction of 
Scheme I are the same as those used in known methods of DNA synthesis, 
e.g., in the phosphoramidite (see Beaucage and Caruthers, Tetrahedron 
Lett. (1981), supra). 
After conversion of the nucleoside or oligonucleotide chain to the 
phosphite triester given by structure X, oxidation- to the corresponding 
phosphate triester (compound XI; Scheme II) may be effected using standard 
techniques, e.g., treatment with aqueous iodine or peroxide in a suitable, 
preferably slightly polar organic solvent such as tetrahydrofuran (THF). 
See, e.g., Horn and Urdea, DNA, supra. 
##STR11## 
Release of the R.sub.1 group is easily monitored, visually or 
colorimetrically, and thus allows for simple and accurate monitoring of 
the overall phosphorylation procedure. 
As illustrated by Scheme II, the base-labile phosphate triester may be 
converted to phosphate salt XII by treatment with a deprotection agent, 
e.g. ammonium hydroxide. This step may proceed concurrently with release 
of the oligonucleotide chain from the solid support where the linkage to 
the solid support is a base-labile one. 
Although phosphitylation and subsequent oxidation and deprotection steps 
(Scheme II) can be carried out in solution, it is preferred that the 
oligonucleotide substrate be bound to a solid support. A wide variety of 
supports may be used, such as silica, Porasil C, polystyrene, controlled 
pore glass (CPG), kieselguhr, poly(dimethylacrylamide), 
poly(acrylmorpholide), polystyrene grafted onto poly(tetrafluoroethylene), 
cellulose, Sephadex LH-20, Fractosil 500, etc. References of interest 
include: Sonveaux, supra; Matteucci and Caruthers, supra, Chow et al., 
Nucleic Acids Res. (1981) 9:2807; Felder et al., Tetrahedron Lett. (1984) 
25:3967; Gough et al., ibid (1981) 22:4177; Gait et al., Nucleic Acids 
Res. (1982) 10:6243; Belagaje and Brush, ibid (1982) 10:6295; Gait and 
Sheppard, ibid (1977) 4:4391; Miyoshi and Itakura, Tetrahedron Lett. 
(1978) 38:3635; Potapov et al., Nucleic Acids Res. (1979) 6:2041; Schwyzer 
et al., Helv. Chim. Acta (1984) 57:1316; Chollet et al., ibid (1984) 
67:1356; Ito et al., Nucleic Acids Res. (1982) 10:1755; Efimov et al., 
ibid (1983) 11:8369; Crea and Horn, ibid (1980) 8:2331; Horn et al., 
Nucleic Acids Res. Sym. Ser. (1980) 7:225; Tragein et al., Tetrahedron 
Lett. (1983) 24:1691; Koster et al., Tetrahedron (1984) 40:103; Gough et 
al., Tetrahedron Lett. (1983) 24:5321; Koster et al., ibid (1972) 16:1527; 
Koster and Heyns, ibid (1972) 16 1531; Dembek et al., J. Am. Chem. Soc. 
(1981) 103:706; Caruthers et al., Genetic Enqineering: Principles and 
Methods, eds. Setlow and Hollaender, Vol. 4, 1982, pp. 1-12, Plenum Press. 
N.Y. 
Phosphitylation using the reagent of Formula III proceeds in a similar 
manner (see Froehler and Matteucci, supra), yielding 
##STR12## 
This compound may then be oxidized and deprotected as described above. 
Phosphorylation using the reagent of Formula IV proceeds according to 
Scheme III and may be deprotected to the phosphate as described. 
##STR13## 
As described in the parent application hereto, depending on the nature of 
the support, different functionalities will serve as anchors. For 
silicon-containing supports, such as silica and glass, substituted alkyl 
or aryl silyl compounds will be employed to form a siloxane or siloximine 
linkage. With organic polymers, ethers, esters, amines, amides, sulfides, 
sulfones, phosphates may find use. For aryl groups, such as polystyrene, 
halomethylation can be used for functionalization, where the halo group 
may then be substituted by oxy, thio (which may be oxidized to sulfone), 
amino, phospho (as phosphine, phosphite or phosphate), silyl or the like. 
With a diatomaceous earth, e.g., kieselguhr, activation may be effected by 
treatment with a polyacrylic acid derivative followed by reaction with 
amino groups to form amine bonds. Polysaccharides may be functionalized 
with inorganic esters, e.g., phosphate, where the other oxygen serves to 
link the chain. With polyacrylic acid derivatives, the carboxyl or side 
chain functionality, e.g., N-hydroxethyl acrylamide, may be used in 
conventional ways for joining the linking group. 
The linking group or chain will vary widely as to length, functionalities 
and manner of linking the first nucleotide. For extending chains, 
functionalities may include silyl groups, ether groups, amino groups, 
amide functionalities or the like, where bifunctional reagents are 
employed, such as diamines and dibasic acids, amino acids, saccharides, 
silanes, etc. 
A number of supports and linking groups which have been reported in the 
literature are shown in the following Table. 
TABLE 
__________________________________________________________________________ 
Support.sup.1 
Linking chain.sup.2 
Terminal group.sup.3 
Reference 
__________________________________________________________________________ 
Silica Si(CH.sub.2).sub.3 NHCO(CH.sub.2).sub.2 CO-- 
DMT-nucleoside 
Mateucci & Caruthers, 
(1980), supra 
Silica Si-(5' att) 3' Ac-Thymidine 
Koster, Tetrahedron Lett. 
(1972), 16:1527 
Silica Si0C(0.sub.2)O-- (5' att) 
Ac-nucleoside 
Ibid. 
CPG LCAA--CO(CH.sub.2)CO-- 
2'-o-O.sub.2 N0CH.sub.2, 
Gough et al., Ibid (1981) 
5' DMT-ribonucleoside 
22:4177 
CPG SiOSi(OEt).sub.2 (CH.sub.2).sub.3 NHCO(CH.sub.2).sub.2 CO-- 
DMT-nucleoside 
Koster et al., Tetrahedron 
(1984) 40:103 
CPG LCAA--CO(CH.sub.2).sub.2 CO-- (5' att) 
2'-0CO-ribonucleoside 
Gough et al., Tetrahedron 
Lett. (1983) 24:5321 
Porasil C 
Si(CH.sub.2).sub.3 NHCO(CH.sub.2).sub.2 CO-- 
DMT-nucleoside 
Chow et al., Nucleic Acids 
Res. (1981) 9:2807 
Kieselguhr - 
N(Me)CH.sub.2 CONH(CH.sub.2).sub.2 -- 
DMT-nucleoside 
Gait et al., Ibid (1982) 
PDMA (COCH.sub.2 NH).sub.2 CO(CH.sub.2).sub.2 CO 
10:6243 
Polystyrene 
CH.sub.2 SO.sub.2 (CH.sub.2).sub.2 OP(Cl0O)).sub.2 
DMT-nucleoside 
Felder et al., Tetrahedron 
Lett. (1984) 25:3967 
Polystyrene 
CH.sub.2 O0C(0)(MeO0)O-- (5' att) 
nucleoside- Belagaje & Brush, Nucleic 
(3'-Cl0-phosphate) 
Acids Res. (1982) 10:6295 
Sephadex LH-20 
OPO.sub.2 (5' att) ribonucleoside 
Koster & Heyns, 
Tetrahedron Lett. (1972) 
16:1531 
Polyacrylamide 
CONH(CH.sub.2).sub.2 NHCO(CH.sub.2).sub.2 CO-- 
DMT-nucleoside 
Dembek et al., J. Am. 
Chem. Soc. (1981) 
103:706 
Fractosil 500 
(CH.sub.2).sub.3 NH(CH.sub.2).sub.2 CO-- 
DMT-nucleoside 
Caruthers et al., Genetic 
Engineering (1982) 4:12 
Polyacryl- 
(CH.sub.2).sub.n NH-- 
ribo- or deoxyribo- 
S. Pochet et al., Tetra- 
morpholide nucleoside dron Lett. (1985) 26:627 
Silica (CH.sub.2).sub.n NH-- 
ribo- or deoxyribo- 
S. Pochet et al., supra 
nucleoside 
CPG (LCAA) 
(CH.sub.2).sub.n NH-- 
ribo- or deoxyribo- 
S. Pochet et al., supra 
nucleoside 
__________________________________________________________________________ 
.sup.1 CPG controlled pore glass / PDMA polydimethylacrylamide 
.sup.2 0 phenyl / Me methyl / Et ethyl / LCAA long chain alkyl amino 
att attachment 
.sup.3 DMT p,pdimethoxytrityl / Ac acetyl / 0 phenyl nucleoside 
intends deoxyribonucleoside / groups indicate Oprotective groups / 3' 
nucleoside attachment, unless otherwise indicated 
5. Use in Purification Method of U.S. Pat. Ser. No. 07/891,789 (now 
abandoned) 
Briefly, the method described in the parent application hereto involves 
synthesis of oligonucleotide chains in such a way that contamination with 
erroneous sequences is minimized. The oligomerization occurs while the 
growing chain remains bound to an insoluble support. After each stage, 
failure sequences are capped and the next monomer added until the sequence 
is complete. Protective groups on the individual monomers, terminal 
blocking groups, capping groups, and linkage to the support are selected 
so as to allow for selectable cleavage. The blocking groups are selected 
so as not to interfere with enzymatic degradation of a sequence lacking 
the terminal blocking group. At completion, the capping group is removed, 
blocking groups which interfere with enzymatic degradation are removed, 
and incomplete sequences lacking the terminal blocking group are degraded 
enzymatically. The oligomers may be retained on the support or removed 
prior to enzymatic degradation of the incomplete sequences. The completed 
correct sequences are then isolated substantially free of sequences having 
errors. 
Thus, the method provides for selective, enzymatic removal of 
error-containing or incomplete oligonucleotides. This is achieved by 
employing terminal blocking functionalities which inhibit an exohydrolase 
from acting on a complete sequence, while the exohydrolase is capable of 
hydrolyzing an unblocked incomplete sequence. The method also employs 
capping functionalities which terminate sequences that have not undergone 
the next stage in the sequential addition, and prior to capping, retain 
the reactive free terminal (5'-OH) functionality. Thus, failure sequences 
terminate at the time of failure and are not continued. 
The reagents disclosed herein may be used in conjunction with the method 
described so as to provide a 5'-phosphate triester blocking group on 
completed oligonucleotide sequences, as described above. Use of the 
phosphate triester as a 5'--O--blocking group avoids degradation of 
complete oligomers by the exonuclease. The 5'-phosphate triester is 
particularly useful with this method, as it is retained during removal of 
the capping groups and during exonucleolytic conditions, and, further, is 
removable without degradation of the oligomer. 
The novel reagents thus provide easy functionalization of the terminal 
5'-hydroxyl of the olignucleotide chain, provide protection of the chain, 
and are readily compatible with automated synthesis of nucleic acid 
sequences. Further, monitoring of the deprotection reaction and thus of 
the completion of oligonucleotide synthesis can be done accurately via a 
simple, colorimetric reaction, i.e., the release of the "R.sub.1 " moiety 
described above.

It is to be understood that while the invention has been described in 
conjunction with the preferred specific embodiment thereof, that the 
foregoing description as well as the examples which follow are intended to 
illustrate and not limit the scope of the invention, which is defined by 
the scope of the appended claims. Other aspects, advantages and 
modifications within the scope of the invention will be apparent to those 
skilled in the art to which the invention pertains. 
EXAMPLE 1 
The reagent 
(2-cyanoethoxy)-2-(2'-4,4-dimethoxytrityloxyethylsulfonyl)ethoxy-N,N-diiso 
propylaminophosphine (see Formula I) was synthesized as follows. 
Commercially available sulfonyldiethanol (65% w/ v in H.sub.2 O) was dried 
by repeated coevaporation with dry acetonitrile to give a viscous oil 
which crystallized on standing. To solid sulfonyldiethanol (10.6 g, 68.6 
mmole) in pyridine (150 ml) was added 4,4'-dimethoxytrityl chloride (16.95 
g, 50 mmole) and the mixture was left stirring in the dark for 18h. The 
reaction solution was then concentrated in vacuo. The residue dissolved in 
ethyl acetate (500 ml) was extracted with 5% aq. NaHCO.sub.3 and 80% 
saturated aq. NaCl and the organic phase was dried over anhydrous Na.sub.2 
SO.sub.4. After removal of solvents the product was purified by silica gel 
column chromatography to give 10.0g of pure 
2-4,4'-dimethoxytrityloxyethylsulfonyl ethanol 1 (TLC, silica in CH.sub.2 
Cl.sub.2 ; R.sub.f =0.015). 
Chloro-N,N-diisopropylamino-2-cyanoethoxy-phosphine 2 (4.6 mmole) was 
added rapidly under argon to a stirred solution of 1 (4.6 mmole) and 
N,N-diisopropylethylamine (DIPEA; 4.6 mmole) in methylene chloride (10 
ml) at 0.degree. C. The solution was allowed to warm to room temperature, 
diluted with ethyl acetate (50 ml) and washed with 80% saturated aq. NaCl 
(2.times.20 ml). The organic phase was dried with anhydrous Na.sub.2 
SO.sub.4 and concentrated by rotary evaporation. The oily product 3 was 
dissolved in acetonitrile and then aliquoted into 1.5 ml septum-sealed 
Wheaton vials each containing 100 micromoles of reagent. The solvent was 
removed by evacuation and the product was stored under argon at 
--20.degree. C. This crude product was used without further purification. 
The dried materials were activated with tetrazole in acetonitrile and 
coupled to solid-supported oligonucleotides. Subsequently the synthetic 
DNA was oxidized with aqueous I.sub.2 under standard conditions and 
deprotected with NH.sub.4 OH at 60.degree. C. This process gives the 
5'-phosphorylated target fragment in quantitative yield. The extent of 
coupling was determined from the absorption spectrum (498 nm) of an orange 
solution produced upon treatment of the oligomer with dichloroacetic acid 
in methylene chloride (5% v/v) prior to deprotection with NH.sub.4 OH. 
EXAMPLE 2 
Enzymatic purification of oligonucleotides in solution: The fragments 
5'-TATCAATTCCAATAAACTTTACTCCAAACC-3, and 
5'-AAGGATCCAGTTGGCAGTACAGCCTAGCAGCCATGGAAAC-3' were synthesized on the CPG 
support (Warner, et al., DNA3, 401 (1984)) The fragments were then 
5'-phosphorylated as described in Example 1. The oligomers were removed 
from the support with NH.sub.4 OH at room temperature, then deprotected 
overnight at 60.degree. C. The solution was evaporated to dryness in a 
speed-vac concentrator. 
The crude product obtained from 2 mg of the support was suspended in 20 
micro 1 of H.sub.2 O to which 50 micro 1 of sodium phosphate buffer, pH 
7.0 containing 0.3 units of spleen phosphodiestetase was added. After 
vortexing the solution was placed at 37.degree. C. for 1 hour. 
Polyacrylamide gel analysis revealed that truncated failure sequences were 
substantially degraded whereas the phosphorylated target fragment was 
protected from hydrolysis. 
EXAMPLE 3 
A phosphorylating agent according to Formula III was prepared as follows. 
The method of Example I was followed through purification of 
2-4,4'-dimethoxytrityloxyethylsulfonyl ethanol. Then, 
chloroN,N-diisopropylamino-2-cyanoethoxyphosphine oxide (4.6 mmole) was 
added to a DIPEA solution as described. The product was isolated by 
precipitation as the barium salt, and used without further purification to 
phosphorylate a completed oligonucleotide sequence as described in Example 
1. 
EXAMPLE 4 
A phosphitylating agent according to Formula IV is prepared by reaction of 
2-4,4'-dimethoxytrityloxyethylsulfonyl ethanol (4.6 mmole) with 
phosphorous acid (4.6 mmole) in pyridine at about 0.degree. C. The 
reaction proceeds in the presence of tosyl chloride as an activating 
agent. The product, 2,4,4'-dimethoxytrityloxyethylsulfonylethoxy 
phosphate, is isolated, e.g., by precipitation, and coupled to synthetic 
DNA via a method similar to that described in Example 1 using 
(CH.sub.3).sub.3 COCl as activating agent. The phosphitylated DNA is 
oxidized with I.sub.2 and deprotected with NH.sub.4 OH to give the 5' 
phosphate. The coupling reaction is monitored as described in Example 1. 
EXAMPLE 5 
Comparison of chemical phosphorylation with enzymatic: The palindromic 
BamHI linker sequence GGATCCGGATCC was synthesized on an automated 
instrument (the Geno-O-Matic) using a solid-supported phosphoramidite 
chemistry (12) One-half of the support was phosphorylated with reagent 3, 
detritylated to check the coupling efficiency and fully deprotected. The 
product was then purified by polyacrylamide gel electrophoresis. The 
second half of the material was deprotected and purified as the 
5,-hydroxyl form which was then 5-phosphorylated with T.sub.4 
polynucleotide kinase and ATP. The PAGE analyses of T.sub.4 DNA ligase 
reactions using the chemically and enzymatically phosphorylated fragments 
showed that both sequences were near fully phosphorylated as evidenced by 
the lack of starting material after ligation