Methods and solvent vehicles for reagent delivery in oligonucleotide synthesis using automated pulse jetting devices

Solvent vehicles and methods of their use in the pulse jet delivery of reagents in automated oligonucleotide synthesis by the solid-phase phosphoramidite method are provided. For pulse jet delivery of the activated monomer, acetonitrile in combination with at least one stabilizer co-solvent is employed as the solvent vehicle, where the stabilizer co-solvent is an inert, organic solvent having a boiling point of at least 100.degree. C. For pulse jet delivery of the detritylation reagent, a mono- or disubstituted methane is employed as the solvent vehicle, where substituents have a molecular weight greater than 36 daltons. Use of the solvent vehicles according to the subject invention provides for reduced reagent precipitation in the pulse jet tubes and improved results.

INTRODUCTION 
1. Field of the Invention 
The field of this invention is solid phase oligonucleotide synthesis. 
2. Background 
Chemical oligonucleotide synthesis has advanced to the point where nearly 
all such synthesis is carried out with highly automated machines. The 
chemistry employed in such machines is known as the phosphoramidite 
solid-phase synthesis method and is described in detail in Itakura et al, 
"Synthesis and Use of Synthetic Oligonucleotides," Annu. Rev. Biochem. 
(1984) 53:323 and Narang et al., "DNA Synthesis," Tetrahedron (1983) 39:3. 
Generally, this method comprises three repetitive steps: (1) a coupling 
step; (2) an oxidation step; and (3) a detritylation step. In the coupling 
step, an activated protonated deoxyribonucleoside 3'-phosphoramidite 
monomer having a dimethoxytrityl blocking group at the 5' position is 
contacted with a solid phase bound nucleoside. The activated monomer is 
typically present in acetonitrile. Contact results in joining of the 
activated monomer to the 5'OH of the solid phase bound nucleoside through 
a phosphite triester group. In the oxidation step, the phosphite triester 
is oxidized to a phosphotriester, usually through contact with I.sub.2. 
Finally, in the detritylation step, the dimethoxytrityl protecting group 
on the newly added activated monomer is removed with a detritylation 
reagent, e.g. di- or trichloroacetic acid. The detritylation reagent is 
typically present in dichloromethane. Through appropriate repetition of 
the above steps, an oligonucleotide of desired length and sequence can be 
synthesized. This method is described in greater detail in U.S. Pat. No. 
4,458,066; as well as in "Oligonucleotide Synthesis-A Practical Approach" 
(Gait, IRL Press)(1984) and Stryer, Biochemistry (1988) pp 123-124. 
Because of the repetitive nature of the above synthesis method, the method 
is particularly suited for automation, in which the above steps are 
automatically performed. A variety of automated oligonucleotide synthesis 
devices capable of synthesizing oligonucleotides according to the 
phosphoramidite method have been developed. See, for example, WO 94/01215. 
In the area of oligonucleotide synthesis, miniaturization of automated 
synthesis devices has become a desirable goal for a variety of reasons, 
e.g. to reduce reagent waste, etc. One class of devices that have been 
developed to meet this goal are pulse jetting devices capable of 
delivering less than 1 .mu.l spots in a reproducible fashion on the 
surface of a substrate. Such devices provide for the possibility of 
producing microarrays of oligonucleotides, which microarrays are finding 
use in new assay applications. See Schena et al., "Quantitative Monitoring 
of Gene Expression Patterns with a Complementary DNA Microarray," Science 
(1995) 270:467-470 and Kozal et al., "Extensive Polymorphisms Observed in 
HIV-1 Clade B Protease Gene Using High-Density Oligonucleotide Arrays," 
Nature Medicine (1996) 2:753-759. 
Despite the promise of automated oligonucleotide synthesis with pulse 
jetting devices, certain obstacles resulting from the chemistry involved 
in the synthesis must be overcome. For example, although acetonitrile 
provides for high efficiency in the coupling reaction, when used in a 
pulse jetting device it tends to evaporate rapidly from the jetting tube, 
resulting in activated monomer precipitation and concomitant plugging of 
the jetting tube orifice. Likewise, dichloromethane can lead to 
detritylation reagent precipitation and plugging of the tube orifice. 
Since reagent precipitation and orifice plugging result in reagent waste 
and time consuming cleaning steps, there is interest in the identification 
of new solvent vehicles for use in reagent delivery through pulse jetting 
devices. The new solvent vehicles should at least reduce, if not 
substantially eliminate, reagent precipitation orifice plugging. Ideally, 
such solvent vehicles should be compatible with the particular synthesis 
reaction being performed, e.g coupling or detritylation, so as not to 
detract from the efficiency of the synthesis. 
Relevant Literature 
U.S. Pat. No. 4,877,745 discloses a reagent fluid dispensing apparatus 
comprising a jetting tube mounted within a cylindrical piezo-electric 
transducer, and reports the use of glycerin in reagent solvent vehicles to 
reduce the problems associated with solvent evaporation. 
SUMMARY OF THE INVENTION 
Solvent vehicles for use in the delivery of reagents during oligonucleotide 
synthesis with an automated pulse jetting device are provided. For pulse 
jet delivery of the activated monomer, acetonitrile is combined with a 
stabilizer co-solvent to produce an anhydrous activated monomer solvent 
vehicle, where the stabilizer co-solvent is an inert organic solvent 
having a boiling point or flash point greater than about 100.degree. C. 
For delivery of the detritylation reagent, a mono-or disubstituted 
methane, where the substituent has a molecular weight greater than 36 
daltons, is employed as the detritylation reagent solvent vehicle, which 
solvent vehicle may further comprise an additional co-solvent. Use of the 
particular solvent vehicles according to the subject invention provides 
for at least reduced reagent precipitation in the pulse jet delivery 
device. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Solvent vehicles, and methods of their use, in the pulse jet delivery of 
reagents in automated oligonucleotide synthesis by the solid-phase 
phosphoramidite method are provided. Specifically, a solvent vehicle 
comprising acetonitrile in combination with an organic stabilizing 
co-solvent having a boiling point or flash point greater than about 
100.degree. C. is employed for pulse jet delivery of the activated 
phosphoramidite monomer in the coupling step. For the detritylation step, 
the detritylation reagent is delivered in a solvent vehicle that provides 
for reduced reagent precipitation, where the solvent vehicle may comprise 
a co-solvent. 
As mentioned above, the subject solvent vehicles are suitable for the pulse 
jet delivery of reagents necessary for oligonucleotide synthesis by the 
phosphoramidite method, as described in the background section above. 
The automated oligonucleotide synthetic devices in which the subject 
solvent vehicles find use are those devices in which the reagents are 
delivered to a substrate by pulse jetting. Generally, such devices 
comprise a reagent tube, e.g. a capillary, having an orifice at one end. 
Associated with the tube in the region of the orifice is a transducer 
means, such as a piezoelectric device or heating element, which forces a 
defined volume of liquid reagent out of the tube through orifice upon 
activation. Pulse jetting devices which are adaptable for use in the 
synthesis of oligonucleotides according to the solid phase phosphoramidite 
method and in which the subject solvent vehicles find use include the 
devices disclosed in WO 94/01215 and U.S. patent application Ser. No. 
08/646,535, the disclosures of which are herein incorporated by reference. 
For the pulse jet delivery of activated monomers in the coupling step 
according to the subject invention, the activated monomer solvent delivery 
vehicles are anhydrous and comprise acetonitrile in combination with at 
least one, usually not more than three, more usually not more than two 
different co-solvents or stabilizers which serve to at least reduce the 
level of, if not substantially inhibit, precipitation of the activated 
monomer in the pulse jet tube. The stabilizer will be an organic solvent 
having a boiling point or flash point of greater than about 100.degree. 
C., usually greater than about 120.degree. C., and more usually greater 
than about 150.degree. C. The molecular weight of the stabilizing solvent 
will generally range from about 70 to 200 daltons, usually from about 80 
to 140 daltons. The organic solvent employed as the stabilizer should be: 
1) be miscible with acetonitrile, 2) not precipitate the solutes, 3) be 
non-reactive with chemistry at hand, 4) reduce the apparent vapor pressure 
of acetonitrile in the vicinity of the orifice (or, in any event, to allow 
for reliable jetting), and, yet, 5) not affect the chemistry on the 
surface, i.e., allow for the linking chemistry to occur and evaporation of 
the droplet before the rinse cycle. The most likely co-solvents which 
satisfies these considerations are solvents that are related structurally 
to acetonitrile, but exhibit lower vapor pressure (as would be expected 
from compounds having high boiling points, or flash points--if boiling 
points are not available). With these considerations in mind, we have 
identified which exhibit improved jetting. These co-solvents and their 
properties are set forth in Table 1. 
TABLE 1 
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ACCEPTABLE CO-SOLVENTS 
Molecular Boiling 
Flash 
CO-SOLVENT ACRONYM Weight Point Point 
______________________________________ 
Trimehtyl- TMP 140 daltons 
197.degree. C. 
phosphate 
Methyl- N-MN 120 daltons &gt;110.degree. C. 
Pyrroleaceto-nitrile 
Methyl- N-MPN 99 daltons 
202.degree. C. 
Pyrrolidinone 
n,n,-Dimethyl 
DMF 73 daltons 
153.degree. C. 
Foramide 
Nitromethane 61 daltons 
101.degree. C. 
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Organic solvents of interest as stabilizers will generally have from 2 to 8 
carbon atoms, more usually 3 to 7 carbon atoms, with 1 or more 
heteroatoms, usually 2 to 5 heteroatoms, where the heteroatoms are 
typically O, N or P, where the stabilizing solvent may be cyclic, where N 
may be an annular atom, and will generally have from 1 to 3 methyl groups 
and no hydroxy groups. Specific stabilizing solvents of interest include: 
amides, such as dimethyl formamide; phosphates, such as trimethyl 
phosphate; pyrroles, such as N-methyl pyrrolidinone and N-methyl 
pyrroleacetonitrile; and the like. 
The amount of stabilizer employed in the activated monomer solvent vehicle 
will be sufficient to reduce the volatility of the vehicle and thereby 
reduce or substantially eliminate reagent precipitation in the jetting 
tube. Generally, the amount of stabilizer will range from about 2 to 10% 
(v/v) of the vehicle, usually from about 2.5 to 10% (v/v) and more usually 
4 to 6% (v/v) of the vehicle. Where either trimethyl phosphate or 
pyrroles, such as N-methyl-pyrrolidinone and N-methyl-pyrroleacetonitrile, 
are employed as the stabilizer, the amount of stabilizer will range from 
about 2.5 to 7.5% (v/v), and will preferably be 5% (v/v) of the co-solvent 
vehicle. Where dimethyl formamide is employed as the stabilizer, the 
amount of stabilizer in the co-solvent vehicle will range from about 2.5 
to 10% (v/v), but will preferably be 5% (v/v) of the co-solvent vehicle. 
For pulse jet delivery of the detritylation reagent according to the 
subject invention, a detritylation reagent solvent delivery vehicle will 
be employed in which reagent precipitation in the jet tube is slowed or 
prevented. The detritylation reagent will typically be zinc bromide or 
trichloroacetic acid. Solvent vehicles finding use include mono- and 
disubstituted methanes, where the substituents will have molecular weights 
greater than about 36 daltons. In the monosubstituted methanes, the 
substituent is preferably a nitro group while in the disubstituted 
methanes, the substituents are preferably halo groups having an atomic 
number greater than 17, where bromine is preferred. Where zinc bromide is 
employed as the detritylation reagent, a solvent vehicle saturated with 
zinc bromide is typically used, where the solvent vehicle is nitromethane 
alone or in combination with at least one additional co-solvent, where the 
volume ratio of nitromethane to co-solvent will range from 8.5:1 to 9.5:1, 
and will preferably be 9:1, where the co-solvent will be a lower alkanol 
or diol, usually having no more than about 10 carbon atoms, more usually 
no more than about 8 carbon atoms, and usually more than 2 carbon atoms, 
where tetraethylene glycol and butanol are preferred. Where 
trichloroacetic acid is employed as the detritylation agent, the solvent 
vehicle will preferably be dibromomethane, where the volume % of 
tricholoracetic acid in the dibromomethane solvent vehicle will range from 
about 0.001 to 0.01. 
The following examples are offered by way of illustration and not by way of 
limitation.

EXPERIMENTAL 
An array of oligonucleotides was synthesized using the pulse jetting device 
described in U.S. patent application Ser. No. 08/646,535, the disclosure 
of which is herein incorporated by reference. For pulse jet delivery of 
the activated phosphoramidite monomer, the following four solvent vehicles 
were employed: 
(a) acetonitrile in combination with 5% (v/v) N-methyl-pyrrolidinone 
(b) acetonitrile in combination with 5% (v/v) dimethyl formamide 
(c) acetonitrile in combination with 5% (v/v) trimethyl phosphate 
(d) acetonitrile in combination with 5% (v/v) N-methyl-pyrroleacetonitrile 
For pulse jet delivery of the detritylation reagent, the following solvent 
vehicles were employed: 
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Detritylation Reagent 
Solvent Vehicle 
______________________________________ 
ZnBr.sub.2 (saturated) 
nitromethane:tetraethylene glycol 
at 9:1 
ZnBr.sub.2 (saturated) 
nitromethane:butanol at 9:1 
Trichloroacetic Acid (0.001 
dibromomethane 
to 0.01%) 
______________________________________ 
With each of the above solvent vehicles listed above, it was found that the 
reagents were soluble in the vehicle, the vehicle did not adversely affect 
the efficiency of the reaction, either coupling or detritylation, and 
reagent precipitation did not occur, even when there were extended periods 
of non-use. 
It is evident from the above results and discussion that solvent vehicles 
particularly suited for the pulse jet delivery of reagents for synthesis 
of oligonucleotides by the solid-phase phosphoramidite method are 
provided. The use of the subject solvent vehicles at least reduces, if not 
substantially eliminates, reagent precipitation in the jetting tubes, and 
yet do not detract from the efficiency of the particular reactions 
occurring during the synthesis. By preventing reagent precipitation, the 
subject solvent vehicles provide for improved results using automated 
pulse jetting devices. 
All publications and patent applications mentioned in this specification 
are herein incorporated by reference to the same extent as if each 
individual publication or patent application was specifically and 
individually indicated to be incorporated by reference. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the appended claims.