FR2544720A1 - IMPROVED INSTALLATION AND PROCESS FOR THE SYNTHESIS OF CHEMICAL COMPOUNDS, ESPECIALLY OLIGONUCLEOTIDES - Google Patents

Abstract

PLANT FOR THE SYNTHESIS OF COMPLEX OLIGONUCLEOTIDES FROM SIMPLER CHEMICAL COMPOUNDS, IN WHICH AT LEAST ONE OF THESE SIMPLER CHEMICAL COMPOUNDS IS CARRIED BY A SOLID MATRIX CONSTITUTED OF PEARLS PLACED IN A REACTION CHAMBER 102, AND IS SUBMITTED MORE FOLLOWING OTHER SIMPLER CHEMICAL COMPOUNDS FLOWING THROUGH THE CHAMBER IN A CURRENT, CAUSING CHEMICAL INTERACTION BETWEEN SIMPLER CHEMICAL COMPOUNDS TO FORM MORE COMPLEX COMPOUNDS. THE PROCESS CONSISTS OF INTRODUCING SEVERAL COMPLETELY ACTIVE NUCLEOSIDES IN A PRIMARY REACTION ZONE, CONTAINING A SOLID SUPPORT, AND ALLOWING THESE NUCLEOSIDES TO REACT SIMULTANEOUSLY ON THIS SOLID SUPPORT TO FORM AN OLIGONUCLEOSIDE.

Description

The appearance of A D N (deoxyribonucleic acid)

  recombinant and genetic engineering as a method of

  important for both basic and applied research has been made possible by the development of several experimental

  much more complex of genetic material and

  living lules that one would have dreamed of there less than ten

  These techniques allow the dissection of the human genome

  individual genes that can be transferred into microorganisms such as bacteria There, genes can be reproduced by bacteria in amounts to define their chemical structure In addition, microorganisms containing the human gene can be

  to produce large quantities of the protein product

  than encoded by the gene, thus allowing the production of

  large quantities of scarce human proteins such as

  interferon, growth hormone and insulin.

  One of the experimental techniques that took

  an essential part of the rapid development of

  Genetics is the chemical synthesis of oligonucleotides of defined structure These oligonucleotides often form an essential part of the process of isolation of a single gene from a library containing tens

  from thousands to millions of genes that can be expressed

  by an individual living cell at any time

  because of the unique chemical structure of A D N, a morbidity

  of a D N having a particular sequence does not

  will know and will only bind to another piece having a complementary sequence, ignoring all the pieces of A D N of a complex mixture having the bad sequences By

  Therefore, small pieces of A D N prepared by synthesizing

  can be used as molecular samples

  to identify much larger pieces of A D N, and even whole genes, which contain the appropriate complementary sequence

  incorporate into the samples can be deduced from the structure

2544720 O

  of the protein encoded by the discomfort by translating

  the genetic code, the protein sequence in sequence

  that of A D N As the genetic code is degenerate, that is,

  say that most of the chemical motifs in the sequence

  protein may be encoded by several groups of

  chemicals of the A D N sequence, one often has to

  synthesize a series of samples of A D N con-

  keeping related but not identical sequences to ensure that the complementary sequence is prepared

  desired gene This imposes severe constraints on

  used to prepare A D N by synthesis To be useful in a routine work, they must be fast, simple, safe and inexpensive, since it is often necessary to synthesize several dozen molecules of A

  D N different to identify a single gene.

  A process for the synthesis of oligonucleotides has been developed by Caruthers et al (M D Matteucci and M H Caruthers, J Amer Chem Soc 103, 3185-3191, 1981, S L Beaucage and M H Caruthers, Tet Lett 22,

1859-1862, 1981).

  These oligonucleotides are prepared by synthesis

  by the progressive addition of monodeoxynucleosides to an

  xynucleoside covalently attached to a silica gel polymeric support The individual stages for each addition involve the condensation of the activated nucleoside under

  form of phosphite (stage 1), the covalent blocking of

  5 'hydroxyl of unreacted nucleoside (stage 2),

  the oxidation of phosphite to phosphate (stage 3) and the elimination

  nation of the 5 'blocking group of the nucleoside added in

  each cycle (stage 4) As soon as the desired number of mono-

  mothers has been added to the growing chain, the oligonucleotide

  The finished acid is removed from the silica support and the other blocking groups are removed from the oligonucleotide by

  treatment with thiophenol and ammonium hydroxide.

  The Caruthers et Coll process presents the

ractérigtiqus-following:

  (1) The oligonucleotides are synthetically prepared

  This multi-stage synthesis is involved in the development of synthetic intermediates. This development simplifies the purification of synthetic intermediates. Excess reagents and most undesired by-products of the reaction are extracted by washing the support with sole

  without removing the growing polymer.

  (2) the phosphites of activated nucleosides are

  intermediates used for oligonucleotide synthesis

  These phosphites react quickly and with a

  These characteristics are ideal for cyclic condensation reactions to be completed on

a polymeric support.

  (3) a series of reagents have been developed

  by Caruthers and Coll for the selective elimination of

  5 'blockers of nucleosides, nucleotides and oligonucleotides

  cleotides These reagents are Lewis acids such as

  Zn Br 2, BF 3, Al C 13, and Ti C 14 These reagents effectively eliminate

  the protective groups of the ethers without degradation

of preformed oligonucleotides.

  (4) The overall synthesis process is extremely

  each addition of monodeoxynucleoside, including

  including all stages of treatment, requires approximately 2 hours

res 1/2.

  (5) yields above 95% are observed with

  each of the activated mononucleosides.

  The polymer support used in the method of Caruthers et al. Is composed of one of four suitably blocked nucleosides, covalently joined to

macroporous silica gel.

  The initial stage of the process of Caruthers et al.

  is the synthesis of activated nucleosides The nucleosides

  all four hold a 5 'blocking group such as di-panisylphenylmethyl ether (dimethoxytrityl, DM Tr) However, any other can be used

  appropriate 5 'protecting group. It is also desirable to

  to protect amino groups on cytosine, the adeno-

  mine and guanine can use benzoyl groups,

  isobutyryl or the like Therefore, 5'-O-dimetho-

  xytrityl-deoxythymidine (DM Trd (T)), 5'-O-dimethoxytri

  tyl-N-benzoyldésoxycytidine (DM Trd (bz C)), 5'-O-dimetho-

  xytrityl-N-benzoyldesoxyadenosine (DM Trd (bzA)), and 5 '-

  0-dimethoxytrityl-N-isobutyryldésoxyguanosine (DM Trd (ib G)),

  have been used as protected nucleosides.

  Protected nucleosides must be activated for coupling Ideally, the activated nucleoside must be stable and easy to handle, and at the same time

  must be highly reactive towards the 5 '-hydro-

  unprotected xyl of the nucleoside to which it will couple.

  These requirements are best met by a stable, partially active phosphite group, which can be converted in situ to the desired reactive compound in a simple reaction. These compounds described by Caruthers et al.

  Coll, include phosphoramidites of nucleosides.

  These can be synthetically prepared by the mode

  following procedure The DM Tr nucleoside (1 mmol) is

  in 3 ml of dry, acid-free chloroform and in diisopropylethylamine (4 mmol) in a 10 ml reaction vessel pre-swept with nitrogen

  Dry (60 sec) is added dropwise a chlorine

  ro-N, N-dialkylaminomethoxyphosphine (2 mmol, the groups

  may be methyl, ethyl, isopropyl

  or the like) with a syringe to the solution under nitrogen at room temperature After 15 minutes, the solution is passed with 35 ml of ethyl acetate in a 125 ml separatory funnel. The solution is extracted 4 times. with a saturated aqueous solution of sodium chloride (80 ml) The organic phase is dried over sulphate

  of anhydrous sodium and evaporated under pressure

  The foam is dissolved with toluene (10 ml) or ethyl acetate (10 ml) and the solution is added dropwise to 50 ml of hexanes.

  (78 C) with vigorous stirring.

  cold board and washed the white powder with 75 ml of cold hexanes (78 C) The white powder is dried

  reduced pressure and stored under nitrogen.

  To begin the synthesis of the desired oligonucleotide, the first monomer must be coupled to a support

  solid, generally a high quality silica gel for

  high-performance liquid film or a tail glass

  the adjusted pore, these two macroporous supports having

  high specific surface area.

  derivative port to allow its attachment to the monomer.

  Caruthers and Coll have described an appropriate procedure

  which involves the reaction of silica with (3-amino)

  propyl) triethoxysilane, the reaction of the trans-

  amine derivative with succinic anhydride, and the blocking of any remaining silanol groups with

  trimethylsilyl chloride Then the DM Tr-nu-

  desired cleoside to the carboxyl group attached to the silica using dicyclohexylcarbodiimide in anhydrous pyridine Residual carboxyl groups remaining at the end

  40 hours of treatment are blocked by treatment of

  p-nitrophenol and then morpholine.

  obtained contains about 50 micro-moles of nucleoside

per gram of silica.

  The stages involved in the fixation of the nucleo-

  next side and subsequent nucleosides are in summary

The following.

  First, the 5 '-DM Tr group of the nucleo-

  side fixed by treatment with an acid (usually Zn Br 2 in nitromethane or trichloroacetic acid in chloroform) Then the silica is washed with solvents to remove the cleavage reagent These solvents include, for example, for Zn Br 2, n-butanol and 2,6-utidine in tetrahydrofuran, then tetrahydrofuran, and for trichloroacetic acid, nitromethane and then methanol, followed by acetonitrile. The desired phosphoramidite is then activated by treatment with 10-20 molar equivalents of tetrazole. and add it to the silica gel to couple it

  to the free 5 '-hydroxyl group of the nucleoside it contains.

  Both the activated nucleoside and the tetrazole are dissolved in a solvent such as acetonitrile As soon as the reaction

  coupling is complete, usually in 10 minutes,

  The excess reagent of the silica with solvents such as tetrahydrofuran and acetonitrile. If desired, the unreacted 5'-hydroxyl groups can be blocked.

  by treatment with acetic anhydride in 2,6-dimethyl

  dine / tetrahydrofuran before solvent washes, although this stage is not essential for synthesis.

  then transforms the phosphorous ester formed by this

  coupling ration in phosphoric ester by oxidation with

  iodine in a mixture of water, 2,6-lutidine and

  Trahydrofuran is then removed the excess iodine solution of the silica by extraction with methanol and then by

  nitromethane to prepare the removal of 5'-DM Tr

boasts.

  The above process is repeated with the addi-

  desired nucleosides until the correct oligonucleotide sequence is obtained. Then the methyl groups contained in the phosphotri

  treatment with thiophenol / triathylamine / dioxane (

  1: 1: 2 in volume) for 45 minutes at room temperature

  This stage is followed by treatment with hydro-

  concentrated ammonium hydroxide at room temperature for 3 hours to hydrolyze the ester uniting the 3 'end of

  the oligonucleotide to the support, a separation of the oligonucleotide

  split cleotide of the support by filtration, and a heating

  filter at 50 C for 12 hours Then purify

  the oligonucleotide by high-performance liquid chromatography

  The remaining 5 '-DM Tr group serves as a marker during chromatography and finally the group -DM Tr is removed by treatment with 80% acetic acid. The 51-DM Tr group can also be removed before purification.

  if the chromatographic method is replaced by an elec-

  trophoresis on polyacrylamide gel.

  One of the main advantages of the Caracci

  thers and coll is its potential speed returns

  can be maintained throughout the process, increase

  the length of the sequence that can be pre-

  prepared, and no intermediate purification stage

  However, he is extremely la-

  borieux, and the automation of the various stages of the process,

  especially those involving repeated coupling

  Additional monomers to the growing chain are desirable. Caruthers and Coll have described a manual apparatus in which the synthesis is carried out.

  this device is not suitable for complete automation.

  because it does not contain in situ formation means of fully activated nucleosides immediately before coupling This is an important limitation, because of the instability in handling these compounds The present invention overcomes this limitation by incorporating a pre-reaction vessel in which the partially activated nucleosides are fully activated

  immediately before introduction into the reaction vessel

  the primary support containing the solid support on which

  the oligonucleotide is prepared synthetically.

  Basically, while Caruthers et al.

  utilize a stepwise reaction process, the present invention differs significantly in that it provides a method in which fully activated nucleosides are reacted simultaneously in a reaction zone comprising the primary reaction vessel containing the

solid support.

  This allows a complete automation of the pro-

  In addition, this makes it possible to use quantities

  This also allows for simple and safe incorporation of any one of the four or four nucleoside monomers to be more expensive than those required by the manual activation technique.

  any coupling step in the synthesis process

  This is not easy to achieve when using the manual activation process, and this is essential when preparing a large series of A molecules.

  D N having similar sequences.

  Another disadvantage of the Caruthers device

  and Coll is using a mechanical pump to move

  the liquid through the reaction vessel containing the silica support This mechanical system is subject to the blockage of the flow caused by the formation of bubbles

  gas or precipitate in the pumping chambers

  me, the pumping action introduces pres-

  which can lead to the fragmentation of the support

  silica and the clogging of the reaction vessel and

  The pump also introduces a dead volume con-

  sizable in the pipes entering the reaction vessel and leaving this volume dead, adding to

  the obligation to prevent the introduction of gas into

  lizations, leads to a significant dilution of the reagents, or to use much larger volumes

  expensive reagents to fill the dead volume.

  Caruthers and Coll has a recycling valve

  ge that allows to pump and repoint the diluted reagents

  through the reactor, but this increases the response times

  and solvent scavenger This invention

  tion uses a pneumatic gas pressure in the

  tanks containing reagents and solvents for

  In addition, it involves the use of dead volume, miniaturized valves, which minimize the movement of these chemicals through the reactor.

  reagent consumption and allow cy-

  coupling time of 15 min, only this mechanical system

  that is not subject to blockage by air bubbles (in fact, it uses an inert gas to purge the largest

  part of the excess reagents of the pipes and

  before completing the purge with solvents) and, as

  it does not use mechanical pulsating pumps, it does not

  no physical destruction of the silica support.

  In brief, the present invention is directed to a new facility for the synthesis of more complex oligonucleotides from simpler chemical compounds, wherein at least one of these chemical compounds more

  simple is carried by a solid matrix comprising several

  beaders placed in a reaction chamber and successively subjected to several other simpler chemical compounds sent through the chamber in a stream,

  causing a chemical interaction between the chemical compounds

  easier to form-the so-called more com-

  plexes Some of the simpler chemical compounds are

  mixed in a pre-reaction container before being

  traveling through the room to give birth to the species

these appropriate reagents.

  Another aspect of the invention is a novel method which includes the introduction into

  a primary reaction zone containing a solid support

  of, several nucleosides fully activated and

  both these nucleosides to react on the solid support for

form an oligonucleoside.

  One of the aims of the invention is to provide a

  new facility for the synthesis of oligonucleotides.

  In particular, one of the aims of the invention

  tion is to provide an improved facility for the

oligonucleotide synthesis.

  One of the aims of the invention is also to provide

  a new process to synthetically prepare an oligo-

  nucleoside by simultaneous reaction, in a reaction zone

  of several inter-reactive nucleosides and complete-

activated.

  These goals and advantages of the invention as well as

  others will emerge from the following detailed description

  made in connection with the accompanying drawings in which:

  Figures 1A and 1B taken side by side represent

  Figure 2 is a sectional view of the primary reactor of Figure 1B, Figure 3 is a partial section on a larger scale of the central portion of Figure 2, and

  FIG. 4 is a section of the pre-reaction container

of Figure 1 A.

  We will now consider the drawings in more detail.

  The installation 10 of the invention is shown in FIGS. 1A and 1B.

  Typically a solvent container 12 com-

  With a valve 14, a pressure regulator 16 and a valve block 18 A pipe 20 provided with a pressure regulator 22 provides an inert gas such as argon The source of inert gas is not shown in the drawings. tank 24 preferably contains a simple chemical reagent such as tetrazole, and it is also

  associated with a valve 26 and the pressure regulator 28.

  The reservoir 30 contains a single nucleoside, partially protected protected OS such as that corresponding

  At the same time, reservoirs 32, 34 and 36

  other nucleoside derivatives, such as those corresponding to thymine, guanine and cytosine, respectively. Tanks 30, 32, 34 and 36 are provided with

  valves 38, 42, 46 and 50, respectively, and

  40, 44, 48 and 52, respectively.

  The container 12 and the reservoirs 24, 30, 32, 34 and 36 have individual flow control valves 54 and flow control valve regulators 56 which allow the tanks to be evacuated into the trap.

waste 114.

  The valve block 18 supplies a pre-reactor 58

  by a pipe 404 A valve 60 and a pipe

  400 evacuate pre-reactor 58 to waste trap 114 prereactor 58 communicates with a valve block 62 through line 402 An inert gas is sent to

  the block of valves 62 via a pipe 64

  66 evacuates to the waste trap 114.

  The mixture of simple nucleosides in the form of pre-reacted compounds moves to a block of valves

* by a pipe 72.

  Tanks 80, 82, 84, 86 and 88 communicate

with the valve block 70.

  The tanks 80, 82, 84, 86 and 88 each have

  valves 90, pressure regulators 92,

  94 Flow Regulator Pops and 96 Regulators

  appropriate flow control valves.

  A primary reactor 98 is placed in an environment

  thermostatic environment 100 and defines a reaction chamber

  102 having an inlet port 104 and an orifice of

exit 106.

  The outlet port 106 is connected to a block of

valves 108.

  The valve block 108 communicates with a collar

  fraction reader 112 The valve block 108

  also with the waste trap 114 either through a

  198, or by a flow controller 200 and a

channel 202.

  The valve blocks 18, 62, 70 and 108 and the

  valves 14, 26, 38, 42, 46, 50, 54, 60, 90 and 94 are

  electrically driven by the control unit 68.

  The primary reactor 98 is shown in detail in FIGS. 2 and 3, it comprises a base 122 carrying a

  sleeve 124 which contains elements 126, 128, 130 and 132.

  Below the base 122 is the block of

  valves 108 discussed above with respect to FIGS.

  res 1 A and 1 B. The elements 122 and 124 are held together

  by an internally threaded collar 134.

  An upper collar 136 holds the element 132

  in the closed position inside a sleeve 124.

  The chamber 102 is filled with macroporous pearls.

  its made up of silica spheres having cavities

  The pearls can also be chemically modified from

very many ways.

  Pre-reactor 58 is shown in detail at

  Figure 4; it comprises a bottle 420, a closure with

  410, a series of connecting tubes 400, 402 and 404 and a sealing washer 440 The tubes 400, 402 and 404 communicate with the valve blocks 60, 62

  and 18, respectively Activation of nucleosides is

  in the cavity 430 inside the vial 420 The mating threaded surfaces 412 and 422 on 410 and 420, respectively, allow compression of the washer 440 on the surfaces 414 and 416 to seal the cavity 430,

  with the exception of connection tubes.

  The valve blocks 18, 62, 70 and 108 are multi-element valves, preferably constructed without dead volume. Valve blocks such as those described by Wittmann and Coll (US Pat. No. 4,008,736), Hunkapiller and Hood 12 (US-P-4,252,769), Hood et al. (US-A-380,109) and Stark (US-A-300,184) are acceptable. The individual valve elements in the blocks are electronically controlled.

  and individually per control unit 68.

  The implementation of the above facility for synthesizing an oligonucleotide consists of

  three basic procedures: introduction of the

  solid port containing the first bound nucleoside in the primary reactor, performing the coupling cycle described below one or more times using one or all of the nucleoside derivatives, at will, at each cycle, and removal of the completed nucleotide linked to the support of the primary reactor, then separation from the support and elimination of the protective groups of the nucleotide The order of nucleoside additions is programmed in the unit of

  command 68 before starting the coupling cycle.

  To introduce the support containing the first bonded nucleoside in the chamber 102 of the primary reactor 98, the primary reactor is partially dismantled by loosening the screw 103 holding the pipe and removing in order, the upper collar 136, the stop ring 121, the Teflon washer, registered trademark for fluorocarbon resins 123, the element 132, the porous Teflon washer 127, the element 130 and, together, the element 128,

  the porous Teflon washer 125 and the element 126

  pulls from the chamber 102 the previously used support material that may be present and throws the washers

  porous Teflon 125 and 127 The reaction is then

  primary unit 98 by replacing, in order, the element 126, a new porous Teflon washer 125, the element 128,

  element 130, a new medium containing the first

  cleoside fixed in chamber 102, the support of Teflon PO-

  127, the element 132, the Teflon washer 123, the

  stop 121 and top collar 136

  mounting by tightening the screw 103 holding the pipe.

  A typical coupling cycle consists of

following ones:

  Stage 1 Duration: 30 seconds Sweep the reaction

  primary carrier with nitromethane (2.5 ml) Effluent

  is sent in line 198.

  Stage 2 Duration: 10 seconds Sweep the reaction

  argon primer to remove nitromethane.

  The effluent is sent into line 198.

  Stage 3 Duration: 30 seconds Sweep the reaction

  primary carrier with 3% trichloroacetic acid

  chloroform (1.5 ml) to effect detritylation.

  The effluent is sent to the fraction collector 112.

  Stage 4 Duration: 10 seconds Sweep the reaction

  primary reactor with argon to remove the acid solution The effluent is sent to the fraction collector 112.

  Stage 5 Duration: 20 seconds Sweep the reaction

  primary carrier with nitromethane (1.5 ml) Effluent

  is sent to the fraction collector 112.

  Stage 6 Duration: 10 seconds Sweep the reaction

  primary reactor with argon to remove nitromethane. The effluent is sent to the fraction collector 112.

  Stage 7 Duration: 20 seconds Sweep the reaction

  primary reactor with methanol (1.3 ml) The effluent is

sent in line 198.

  Stage 8 Duration: 10 seconds Sweep the reaction

  primary reactor with argon to remove methanol.

  The effluent is sent into line 198.

  Stage 9 Duration: 30 seconds Put the reservoirs

  see nucleosides under argon pressure.

  Stage 10 Duration: 15 seconds Sweep the reaction

  primary reactor with acetonitrile (1.1 ml) Effluent

  out of the primary reactor is sent into the pipeline

  198 Send nucleoside phosphoramidite into

  acetonitrile (0.2 ml) in the prereactor.

  Stage 11 Duration: 5 seconds Sweep the reaction

  primary reactor with acetonitrile (0.4 ml) Effluent

  out of the primary reactor is sent into the pipeline

  198 Sweep the prereactor with argon for

  Serum nucleoside phosphoramidite in the prereactor.

  Stage 12 Duration: 30 seconds Put under

  pressure the tank the tetrazole.

  Stage 13 Duration: 20 seconds Sweep the reaction

  primary reactor with acetonitrile (1.4 ml) Effluent

  out of the primary reactor is sent into the pipeline

  198 Send tetrazole into acetonitrile (0.4 ml)

in the pre-reactor.

  Stage 14 Duration: 5 seconds Sweep the reaction

  primary reactor with acetonitrile (0.4 ml) Effluent

  primary reactor is sent into line 198.

  Sweep the prereactor with argon to push

trazole in the pre-reactor.

  Stage 15 Duration: 15 seconds Sweep the reaction

  primary reactor with acetonitrile (1.1 ml) Effluent

  primary reactor is sent into line 198.

  Bubble argon in the pre-reactor to mix

  nucleoside and tetrazole solutions and

  kill nucleoside activation for coupling.

  Stage 16 Duration: 10 seconds Sweep the reaction

  primary reactor with argon to remove acetonitrile.

  The effluent is sent into line 198.

  Stage 17 Duration: 2 seconds Put the prereaction

pressure vessel with argon.

  Stage 18 Duration: 360 seconds Hunt the nu-

  activated cleoside of the pretreater through the primary reactor

  mayor with argon sent into the prerogative This

  operation performs the coupling of the activated nucleoside to

  cleotide attached to the support present in the primary reactor

  mayor Effluent leaving the primary reactor is sent

  through the flow regulator 200.

  Stage 19 Duration: 5 seconds Sweep the precondition

  actor and the primary reactor with argon to eliminate

  activated nucleoside Effluent leaving the primary reactor

  re is sent in line 198.

  Stage 20 Duration: 10 seconds Send credit

  tonitrile (1.5 ml) in the prereactor.

  Stage 21 Duration: 30 seconds Remove acetonitrile

  trile the prereactor through the primary reactor with this argon sent into the prereactor The effluent leaving the

  primary reactor is sent into line 198.

  Stage 22 Duration: 10 seconds Sweep the reaction

  primary reactor with acetonitrile (0.7 ml) Effluent

  out of the primary reactor is sent into the pipeline

  198. Send acetonitrile (1.5 ml) to the

reactor.

  Stage 23 Duration: 25 seconds Sweep the reaction

  primary (1.8 ml) with acetonitrile Effluent

  out of the primary reactor is sent into the pipeline

  198 Sweep the prereactor with argon to elimi-

  to mine acetonitrile Effluent leaving the prereactor is

sent in line 66.

  Stage 24 Duration: 10 seconds Sweep the reaction

  primary reactor with argon The effluent is sent

the line 198.

  Stage 25 Duration: 60 seconds Sweep the reaction

  primary body with iodine / water / 2,6-lutidine /

  trahydrofuran (2.0 ml) to effect the oxidation of phosphorus

  Phosphate Phosphate The effluent is sent into the canalisation

198.

  Stage 26 Duration: 10 seconds Sweep the reaction

  primary reactor with argon to remove the solution

  iodine The effluent is sent into line 198.

  Stage 27 Duration: 60 seconds Send metha-

  (3.0 ml) through the primary reactor The effluent is

sent in line 198.

  Stage 28 Duration: 10 seconds Sweep the reaction

  primary reactor with argon The effluent is sent

the line 198.

  When the coupling cycle described above has

  repeated the number of times desired, the oligonucleotide ter-

  substrate is removed from the primary reactor for

  separating the support and the

  The removal of the primary reactor is effected

  killed by repeating the procedure described above to introduce a new silica support into the reactor

  The operation of splitting and unblocking is

  subsequently performed manually out of the apparatus 10 by a

  process such as that described by Caruthers et al.

  As an alternative to the ordinary coupling cycle, mention may be made of the use of Zn Br 2 (saturated solution in nitromethane> rather than the trichloroacetic acid solution for the detritylation stage In this case, the detritylation must be pushed to at 10 to 20

  Another variant is to send more than one

  cleoside activated in the primary reactor during

  This is done by sending two, three or

  four nucleosides partially activated in the prereaction

  (using 1/2, 1/3 or 1/4 normal nucleotide sending time, respectively), adding the tetrazole to the nucleoside mixture, and repressing the mixture of

  nucleosides activated through the primary reactor.

  The degree of completion of the coupling reactions can conveniently be monitored by collecting, at each cycle, the effluent from detritylation and subsequent solvent scavenging steps and measuring the 498 mu absorbance of the dimoxytrityl dim cation at the spectrophotometer. The

  relative amounts of absorbance in each of the tubes suc-

  collector cessation can be used

  to calculate a coupling efficiency per cycle

  Typical factors are 95% or more.

Claims (8)

  1 Advanced facility for the synthesis of complex oligonucleotides from chemical compounds   simpler, characterized in that at least one of these   The simplest chemical poses are carried by a so-   lide comprising several beads housed in a reaction chamber (102) and is successively subjected to several other simpler chemical compounds sent through the   chamber in a current, causing a chemical interaction   that between the simpler chemical compounds to form said more complex compounds, in that a pre-reactor (58) is provided, together with and in fluid communication with the reaction chamber (102), and in that partially activated nucleosides are fully activated   immediately before their introduction in the reaction chamber. tion (102).   2 Installation according to claim 1, characterized   characterized in that a series of reservoirs (80, 82, 84, 86, 88) for containing reagents and solvents are provided   upstream of the reaction chamber (102) and in communication with   fluid with it, and that pneumatic means   to apply a gas pressure are provided in Q-   with these tanks to make a displacement   of these materials through the reaction chamber tion (102).   3 Installation according to claim 2, characterized   characterized in that a series of reservoirs (12, 24, 30, 32,   34, 36) for containing reagents and solvents are also   provided upstream of the pre-reactor (58) and in communi- cation of fluid with it.   4 Apparatus according to claim 3, characterized   characterized in that valve blocks (18,62,7-0) are provided in   mounting the pre-reactor (58) and between the pre-reactor (58) and the reaction chamber (102) to regulate the flow of fluids, and in that said valve blocks have no dead volume. Plant according to claim 4, characterized in that said beads consist of   spheres of macroporous silica containing cavities.   6 Apparatus according to claim 1, characterized   characterized in that a valve block (108) is also pre-   seen downstream of the reaction chamber (102) and in communica-   fluid with it, this valve block communicating   further with a fraction collector (112).   7 Apparatus according to claim 6, characterized   characterized in that the macroporous silica spheres are supported by a porous fluorocarbon resin sheet   forming the bottom of the reaction chamber (102).   8 Process of introducing into a zoo   primary reaction containing a solid support,   nucleosides fully activated and allow these nucleosides to react simultaneously on the support for   forming an oligonucleoside, characterized in that   Partially activated osides are fully activated in a pre-reaction zone just prior to their introduction into the primary reaction zone.   Process according to claim 8, characterized   in that the solid support carries a nucleotide and the   so-called active nucleosides couple to this nucleotide.