CA1234060A - Process for the preparation of oligonucleotides - Google Patents

Process for the preparation of oligonucleotides

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Publication number
CA1234060A
CA1234060A CA000461270A CA461270A CA1234060A CA 1234060 A CA1234060 A CA 1234060A CA 000461270 A CA000461270 A CA 000461270A CA 461270 A CA461270 A CA 461270A CA 1234060 A CA1234060 A CA 1234060A
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Prior art keywords
nucleotide
nucleoside
groups
group
carrier
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CA000461270A
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French (fr)
Inventor
Hubert Koster
Nanda D. Sinha
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Proligo LLC
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Individual
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/26—Amides of acids of phosphorus containing P-halide groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Abstract of the disclosure The invention relates to a process for the prepa-ration of oligonucleotides by the following steps:
reaction of a nucleoside with a phosphine derivative, reaction of the nucleotide derivative thus obtained with a nucleoside bonded to a polymeric carrier, oxidation of the carrier-bound nucleoside-nucleotide thus obtained with formation of phosphotriester groups, blocking of free primary 5'-OH groups, elimination of a protec-tive group from the terminal 5'-OH group, where approp-riate single or multiple repetition of the abovemen-tioned steps to introduce further nucleoside phosphate or oligonucleoside phosphate units, and cleavage of the nucleoside-carrier bond and, where appropriate, elimina-tion of all protective groups present in the oligonucleo-side phosphates. The phosphine derivative used is a compound of the general formula III

Description

~L'23~06U

Descript;on The ;nvent;on relates to a process for the prepar-at;on of ol;gonucleot;des of the general formula I

~
~7 R~ _ 0 _ 0~ _ - 0 ~B ' ( I) o 0 R~
O--p--o--n )~

0~ ~

1 r~
The oligonucleotides prepared according to the invent;on have defined sequences and can be used as spec;f;c pr;mers and probes and are of great ;mportance for the synthes;s of complete genes (Arzne;mittelforschung
2~ 30, 3a, 548, (1980)).
Accord;ng to the most recent state of the art, ol~gonucleot;des are prepared by either the phosphate y or phosphite triester method using polymer;c carr;ers (Nachr. Chem. Tech. Lab. 29, 230 (1981)). In order to be able to construct def;ned sequences, ;t is necessary for the ;ndividual units (nucleosides or nucleot;des) to be ,~jr~

~L~34~:)60 provided with suitable protect;ve groups. In this con-text, base-labile acyl groups are generally used for the protection of the exocyclic amino groups on the hetero-cycl;c nucleobases, and a base-labile ester bond ;s used to attach the oligonucleot;de cha;n to the polymer;c car-r;er in a customary manner, and acid-labile trityl ether groups are used to protect the primary 5'-OH group. The phosphate protective group used ;n the phosphate triester method ;s customar;ly e;ther the 2-chlorophenyl or the 4-chlorophenyl group~ w;th an ester-~ype bond, wh;ch can only be removed by attack of a base or a nucleoph;le on the phosphorus atom. This type of step is ;nherently un-desirable since it involves the risk of cleavage of the internucleot;de phosphate ester bond. This risk has been greatly reduced by the use of oximate an;ons (Tetrahedron '.'i ~5 - la -;, ~ 23~
Lett. 19, 2727 (1978)), although these also attack the phosphorus atom in an unclesired manner in the crucial step and, moreover, have the disadvantage ~hat a relatively small amount of des;red ol;gonucleotide is contam;nated with very large amounts of ;nvolat;le salts wh;ch are d;ff;-cult to extract. Th;s not only makes the ~orking up and subsequent pur;f;cat;on of the synthes;zed ol;gonucleot;de diff;cult but also leads to considerable mater;al losses.
In the phosph;te tr;ester method, the methyl group w;th an ester-type bond ;s customar;ly used as the phos-phate protect;ve group which can be removed by attack of a nuçleoph;le on the methyl C atom (J. Amer. Chem. Soc.
99, 3~26 (1977)). Since attack on the P atom is avoided, there is likew;se avoidance of the r;sk of cleavage of the ;nternucleot;de bond. The nucleophile customar;ly used ;s th;ophenol/tr;ethylam;ne, wh;ch are unpleasant to man;-pulate and, moreover, lead to ;nvolatile compounds which are diff;cult to extract and wh;ch, as ment;oned above, both make work-up d;ff;cult and lead to cons;derable mate-r;al losses.
Although the actual synthes;s of ol;gonucleotidesby the sol;d phase/phosph;te or phosphate tr;ester method takes place very eff;c;ently and rap;dly, the preparation of ol;gonucleot;des of deF;ned sequence remains very t;me-consum;ng. Th;s ;s pr;mar;ly due to the problems of thesubsequent work-up and pur;f;cat;on which take up a mul-t;ple of the actual synthes;s time. The process of the invention operates at this point and provides ;n th;s connection a cruc;al techn;cal improvement.

` ! 2 ~34~

Accordingly the present invention provides a pro-cess for the preparation of oligonucleotides of the general formula I

~/
5 ~1 R~ _ _ O-p _o~B (I) O R~

O - p - O -- ~ 8 _ 1~ r~
OH R~

in which ~ denotes a nucleobase, R1 denotes hydrogen, hyd-roxyl or hydroxyl wh;ch ;s protected by the protect;ve groups customary ;n nucleot;de chemistry, and r denotes an ;nteger from 1 to 2no, comprisirlg the steps of:
a) reacting nucleoside of the general formula II

R

0~

25 ;n wh;ch R1 is defined as above, and RZ denotes a pro-tect;ve group customary ;n nucleot;de chem;stry and ~' de-notes the nucleobase ~ protected, where appropr;ate, by the protect;ve groups customary ;n nucleot;de chem;stry, with a n 2 a 1~406~) phosphine derivative of the general formula III

R3 - ~ - P ~ (III) L
in which R3 is a protective group which can be eliminated, and X
and L are groups which react with hydroxyl groups in the sugar moieties of the nucleotides or nucleosides, in the presence of a base to thereby form a nucleotide derivative as further defined below, b) reacting the nucleotide derivative of the formula IV obtained in step a) and represented by the formula IV:

~I
_ ~ (IV) o R~

in which B', Rl, R2, R3 and L are as defined above, with a nucle-~
oside, of the general formula V, bound to a polymeric carrier H~ B' 30~/ ~
O ~ (V) O~

- 2b ~
, .. , .. .. .... _ .. _ ~2340~0 in whi.ch B' and Rl are as defined above and C denotes the poly-meric carrier c) oxidizing the carrier-bound nucleoside-nucleotide of the gen-eral formula VI, obtained in step b) and represented by the for-mula:

R~0 -~ 0 ~o ~1 '
3 I B tVI) R - 0 - P - ~ 0 0 = C ~

in which B', Rl, R2, R3 and C are as defined above, wi-th forma-tion oE phosphotriester groups, d) blocking free primary 5'-OH groups, which have not been re-acted in the reaction according to step b), with permanent pro-tective groups customary in nucleotide chemistry;
e) eliminating the protective group R ;
~) when required repeating steps a) to e) to introduce further nucleoside phosphate or oligonucleoside phosphate units; and g) cleaving the nucleoside-carrier bond and when required elimi-nati.ng protective groups present in the o:L:l.gon-lcl~os.tdc phos-pha-tes \ - 2c -9) ~234060 wh;ch process compr;ses using in step a) as the phosphine derivative of the general formula III a compound in which R3 denotes a group of the formula VII

Y Y
( V I I ) Z -- C -- C --H Y

in which the groups Y, wh;ch can be identical or different, represent hydrogen, methyl and/or ethyl and ~ represents an electron-attracting group, where, in the phosphine der;vat;ve of the formula III, X ;s chlorine, brom;ne, CN or SCN and ~

is CN or SCN, a secondary am;no radical of the formula tVIII) - NR2 (VIII) where the groups R~ are primary, secondary or tertiary alkyl rad;cals hav;ng 1 - 10 carbon atoms, or together form a cycloalkyl radical having 5 - 7Icarbon atoms, wh;ch can con-tain one or two n;trogen, oxygen or sulfur atoms as hetero-atoms, or are ;m;dazole, tr;azole, tetrazole, 3-n;tro-1,2,4-tr;azole, th;azole, pyrrole, benzotr;azole, optionally sub-st;tuted ;n the phenyl mo;ety, or benzohydroxytr;azole, op-t;onally subst;tuted in the phenyl moiety.
` 25 - 2d -~234~6S~ , The invention also provides a protected nucleotide hav-ing the formula:

O ~
I

~o-~-L

where, sl is a protected nucleobase; R1 is H, OH, or a hydroxyl group which is protected by a protective group customary in nucleotide chemistry; R2 iS a protective group;

R is - cl - C - z L is CN, SCN, or NR2; R4 is a primary, secondary or tertiary alkyl radical having 1 - 10 carbon atoms, or where l~2 is a cy-cloalkyl radical having 5 - 7 carbon atoms optionally having one or two nitrogen, oxygen or sulfur atoms as heteroatoms; Y is H, CH3, or CH2CH3; and - 2e -~ .

~L~3~0Çi~

Z i5 a halogen, CN, NO2, phenylthio, phenyl-sulfoxy, or phenylsulfonyl, where the phenyl radicals, may be substituted in the o, o, or ~
positions with a halogen, CN, or NO2 radical, or where the group y -- C -- Z
H
may be replaced by CF3, CC13, or CBr3.

The invention also provides a methocl of preparing oligonucleotides of the general formula:

~< `J

o ~-¦ 0= p -o ~ a OR R

wherein B is a nucleobase, Rl is hydrogen, hy(lroxyl c-r hydroxyl wllicl~ is prot~cted by nucLeosiclc protective groups, and n denotes an integer from 1 to 200, comprising the steps of a) rcacting a nucleotide phosphite represented by the formula:

- 2f -~234~fi~
B' o I

N C C~llCll~O - P--L
wherein B' is a protected nucleobase B, R
is as defined above, R2 is 4,4' dimeth-o~ytrityl or 4,4',4'' trimethoxytrityl; and L is N,N-dimethylamino, N,N~diethyl-amino, N, N-diisopropylamino, or N-morpholino, with a nucleoside bound to a polymeric carrier, 10of the general formula:

~o '~`l O R
15 o= C - ~

wherein s' and Rl are as defined above and C
represents the polymeric carrier to produce a carrier bound nucleoside-nucleotide of the 20formula: .

~ O ~J

~' ~cc;~cl~-O-P--a~o~
, o R~
~, O = C--(3 ~ 2g -1~340t~

wherein B', Rl, R2, R3 and C are as defined above, b) oxidiæing the carrier bound nucleo-side-nucleotide;
c) blocking free primary 5'-OH groups, which have not been reacted in the reaction of step a), with permanent protective groups;
d) eliminating the protective group R2;
f~ repeating steps a) to d) to introduce further nucleoside phosphate or oligo-nucleoside phosphate units; and g) cleaving the nucleoside-carrier bond and optionally eliminating protective groups present in the oligonucleoside phosphates.

~ - 2h -o~ ~

In order to obtaln compounds of the formula ~, In whlch B denotes a nucleobase, for example adenlne (A), guanlne ~G), cytoslne (C), thymlne (T) or uracll (U) or thelr analogs, and R1 denotes hydrogen, hydroxyl or hydroxyl whlch Is protected by the protectlve groups customary In nucleotlde chemlstry, and n denotes an Integer from 1 to 200, accordlng to the Inventlon n varlety of deflned reactlon steps are carrled out, as follows:

a) Reactlon of a nucleoslde of the generai formula I~.

R1 of the general formula ~ can be hydrogen; In thls case the compounds of the formula I are ollgoedeoxy-nucleotldes.
The group R1 can also be hydroxyl or hydroxyl whlch Is, where approprlate, protected by the protectlve group customary In nucleotlde chemJstry. Examples of protectlve groups of thls type are trltyl, monomethoxytrltyl and dImethoxytrltyl, acyl, for example acetyl, benzoyl; tetrahydropyranyl, methoxytetrahydro-pyr~nyl, o-nltrobenzyl ~nd sllyl ethcr3, suc~ ~s~ for ox~mplo, t-butyldlphenylsllyl ethers. A general revlew of the protectlve groups customary In nucleotlde chemlstry to be found In, for example, Tetrahedron 1981, pages 363-369, Lleblgs Ann.Chem. 1978, 839-850, and Nuclelc Aclds Research, Symposlum Serles No. 7, 1980, 39-59.

R2 Is llkewlse a protectlve group customary In nucleo-tlde chemlstry accordlng to the above-mentloned publlcatlons, preferably the acld-lablle 4,4'-dlmethoxytrltyl or 4,4',4"-trlmethoxytrltyl group. B' can llkewlse have a protectlve group customary In nucleotlde chemlstry accordlng to the above-mentloned prlor publlc~tlons.

The nucleoslde of the formula rr Is reacted accordlng to the Inventlon wlth a phosphlne derlvatlve of the general formula ~

In the general formula, X denotes chlorlne~ bromlne, CN

or SCN; L denotes chlorlne, bromlne, CN, SCN or an amlno radlcal of the formula -NR4 (formula Vlll), where the group R4 denote prlmary or secondary or tertlary alkyl radlcals havlng 1-10 carbon atoms, or together denote a cycloalkyl radlcal havlng 5-7 carbon atoms, optlonally wlth alkyl branches, and/or can contaln on~ ~r tw~ nltr~Qn, ~xya~n nnd~or sulfur atoms ns hetero-atoms.
The group L can also form a reactlve heterocycllc radlcal, the Imldazolyl, trlazolyl, tetrazolyl, 3-nltro-1,2,4-trlazolyl, thlazolyl, pyrrolyl, benzotrlazolyl (optlonally wlth substltuents In the phenyl molety) or benzohydroxytrlazolyl (optlonally wlth substltuents In the phenyl rlng) and the llke.

R3 In the phosphlne derlvatlve of the general formula (111) Is, accordlng to the Inventlon, a group of the general formula Vll, whlch can be removed wlth the ald of bases by ~ -elImlnatlon and In whlch Y denotes hydrogen, methyl or ethyl. Z
represents an electron-attractlng group, for example, halogen, such as fluorlne, chlorlne or bromlne, CN or N02. Z can also denote phenyl, phenylthlo, phenylsulfoxy or phenylsulfonyl, It belng posslble for the phenyl radlcals to be substltuted In the o, o'-posltlon and/or p-posltlon wlth halogen, CN or N02. It Is also posslble for one of the groups CF3, CCI3 or CBr3 to replace the group Z C

The reactlon accordlng to step a ta~es place In the presence of an organlc base.

b) Reactlon of the nucleoslde-phosphorous acld derlvatlve, of the formula 1V, obtalned In step a.

~.23~06~

The reactlon of the compound accordlng to formula IV Is carrled out wlth a nucleoslde of the general formula ~ whlch Is bound to a polymerlc carrler. It Is posslble to use soluble or Insoluble, that Is to say crosslInked, polymerlc carrlers, for example modlfled slllca gel, glass, especlally "controlled pore ~In.~ oly~t~r, polynmld~, polyvlnyl nlcohol, polyslloxane, polystryene or the llke. Ester bonds are sultable and preferred for the attachment between the carrler and the nucleoslde, Inclu-dlng those derlved from the levullnyl or ~ -benzoylproplonyl rad-Ical; the latter ester bonds can be cleaved wlth hydrazlne underneutral condltlons. The acld-lablle trltyl ether bond, optlon-ally wlth substltuents In the phenyl rlngs, Is also sultable as a method of attachment, comPare Lleblgs Ann.

1~

~ \

~23~060 Chem. 197~, 959.
c) Oxidation of the carrier-bond nucleotide-nucleo-side, of the general formula VI, obtained ;n step b.
Oxidation leads to a phosphate group; this can be S carried out with, for example, iodine/H20, H202 or organ;c perac;ds or, ;n general, by ox;dation by ;ntro-duction of 0, S or Se.
d) Blocking of free primary 5'-OH groups which have not been reacted in the reaction according to step b (in the product of the formula V).
These free hydroxyl groups are blocked with a per-manent protective group, for example by reaction with acetic anhydride.
e) Elimination of the protective group(s) R2.
The el;m;nat;on ;s carr;ed out us;ng, for example, a proton;c ac;d or Lew;s ac;d, such as ZnBr2 or d;alkyl-alum;nulm chlor;de, when R2 represents a tr;tyl group or a methoxy derivative thereof.
f) Introduction of further nucleoside phosphate or 2û ol;gonucleos;de phosphate units.
Steps a - e can be repeated to introduce at least one nucLeoside phosphate mo;ety. Of course, when oligo-nucleos;de phosphate units are employ~d, the chains are lengthened by more than one nucleoside phosphate unit.
9) EL;m;nat;on of all protective groups.
Th;s elimination can be carried out in such a manner that, using aqueous ammon;a, in one step the N-acyl groups on the heterocyclic bases, the ester bond be-tween the oligonucleotide and the carrier (the latter can, where appropriate, also be cleaved with hydrazine under neutral conditions) and the phosphate protective group are eliminated by ~~elimination in accordance with the gene-ral scheme 1 at the end of the description. An oligo-nucleotide having only a 5'-term;nal trityl protective group is then obtained, and this can be purified directly in a manner known per se, after removal of the volatile base ~ammonia), by high-pressure liquid chromatography (HPLC) on reverse phase material.
The intermediates of the general formula IV accord-ing to claim 1 are new compounds. They are in the form of very stable compounds which can be prepared in the pure form and are easy to manipulate but nevertheless are very re-active in the sense of forming ;nternucleotide bonds.
The use of R3 as a protective group which can be removed by bases via ~-elimination makes it possible for the first t;me to eliminate all the protective groups, apart from the 5'-trityl group, in one step where, in an advan-tageous manner, by the use of volatile bases the desired oligonucleot;de ;s contam;nated with fore;gn mater;als to only a very small extent and thus directly afterwards can be purif;ed by reverse phase HPLC due to the hydrophobic 5'-tr;tyl group which ;s still present.
A further advantage of the process of the ;nven-tion results from the fact that, due to the removal of the protect;ve group by ~-elimination, no attack on the P-atom takes place and thus none of the newly formed inter-nucleotide bonds can be cleaved during the deprotection.
Thus, the process of the invention takes very much less ~3~060 t;me and leads to overall purer products than do the pro-cesses hitherto available.
The ;nvention is ;llustrated in detail below by means of examples, ~he phosphine derivatives used being those in which R3 is a l3~cyanoethyl group. Details of the reaction and physical characteristics of the compounds prepared can be seen in schemes 2 and 3, Table 1, and Figures 1 - 7 at the end of the description.
Example 1:
Preparation of phosphine derivatives of the general formula III:
-Cyanoethyl phosphoramidochloridite:
A general summary of the reaction can be seen in scheme 2.
Apart from some improvements, d;chloro- -cyano-ethoxyphosph;ne (1) is prepared as in Can. J. Chem. 58, 2686 (198û):
300 ml of ether and 79.0 9 (1 moL) of pyr;dine are added through a dropping funnel to 137.5 9 (1.0 mol) of PCl3 ;n a three-neck flask; the m;xture is cooled to -78C
under argon. Then a solution of 71.0 9 ~1 mol) of ~-cyanoethanol ;n 150 ml of dry ether is added dropw;se over the course of 1 to 1.5 hours~ The cooling bath is removed;
stirring is cont;nued at room temperature for a further 3 hours (where necessary, another 3ûO ml of ether are added in order to ensure better st;rrabil;ty). The stirrer and dropping funnel are removed under argon; the mixture is stored at 0C overnight. The solid salts are re-moved under argon; the precipitate is washed twice with ~Z3~060 75 ml of ether each time. The combined organic phases are concentrated in vacuo; the residue is finally dis-tilled in vacuo: boiling point 70 - 75C/0.4 mm Hg.
-Cyanoethyl phosphoramidochloridite (3~:
S A solut;on of 17.2 9 (0.1 mol ) of ~-cyanoethyl phosphorodichloridite (1) in 60 ml of ether is added drop-wise, over the course of 1 to 1.5 hours, to a solution of the N-trimethylsilylated secondary amine tO.1 mol) or secondary am;ne (0.2 mol) ;n 30 ml of ether at -20C
under argon. After st;rr;ng at room temperature for 20 hours, the am;ne hydrochloride is removed; the remaining solution ;s concentrated. The res;due is finally distilled in vacuo in a short-path distillation apparatus.
The physical properties of the compounds thus ob-ta;ned are summar;zed in Table 1.
F;gures 1a, 1b and 1c show 31P NMR spectra ofthree different l~cyanoethyl phosphoramidochloridites.
The N-morpholine derivative is too unstable to heat for distillation to be possible. Nevertheless, the preparation is so pure that the residue can be usecl di-! rectly for the preparation of the activated nucleoside derivatives. The purity ;s usually greater than ~5X
according to the 31p NMR spectra.
Nucleos;de ~-cyanoethyl phosphoram;dites:
The preparation of the appropr;ately protected nucleoside ~-cyanoethyl phosphoramidites can be seen in scheme 3.
The synthesis ;n analogy to Tetrahedron Lett. 22, 1859 (i981), with some improvements, provides good yields.

3.0 mmol of the N-protected 5'-dimethoxytritylated deoxynucleoside are dried azeotropically using THF/toluene~
dissolved ;n 15 ml of dry THF, and 12.0 mmo~ of N,N,N-d;-isopropylethylamine are added. 6.0 mmol of the ~-cyano~
ethyl phosphoram;dochloridite are added dropwise to the solution under argon, with v;gorous stirring~ over the course of 2 minutes. After a short time (2 to 5 minutes), the amine hydrochloride precipitates out. The suspension is st;rred for a further 30 to 40 minutes. The amine hydrochloride is filtered off under argon and thoroughly washed with dry THF (10 to 15 ml). The entire organic phase is concentrated and d;ssolved in argon-saturated ethyl acetate (100 ml). The organic phase is extracted twice with 50 ml each time of argon-saturated 10% aqueous sodium carbonate solution. The organic phases are dried with sod;um sulfate and evaporated under reduced pressure to give a foam. The foam is dissolved in a little ethyl acetate or toluene and precipitated in n-hexane at -78C.
The activated nucleosides are stable for several months when stored at -20C under argon.
Figure 2 shows the 31p NMR spectrum of one of the activated deoxynucleosides.
Synthesis of d~CGGTACCG) 100 mg of "controlled pore glass" tCPG) loaded with a total of 8 umol of N-isobutyryldeoxyguan;ne (com-pare Tetrahedron Lett. 24, 747 t1983)) are consecutively condensed w;th the 5'-d;methoxytr;tylated N-acylated ~-cyanoethyl N,N-diisopropylphosphoramidites of the deoxy-nucleosides C, C, A, T, G, G and C, in each case 20 to 25 1;~34060 equivalents of the phosphoramidite in acetonitrile being act;vated with 75 - 80 equivaLents of sublimed tetrazole.
The condensations are complete within 30 minutes at the most; the coupling yield is greater than 94%. After each condensation, oxidat;on w;th I2lHzO and block;ng of un-reacted 5 -OH groups with acet;c anhydride are carried out.
Then the d;methoxytr;tyl group ;s elim;nated either w;th 3% tr;chloroacetic acid in nitromethane/1% methanol or ZnBr2~n;tromethane/1X H20.
The overall y;eld of the protected octanucleotide at the end of alL condensat;on steps ;s 55% based on carr;er-bound deoxyguanosine.
Complete deprotection and cleavage off from the carr;er ;s ach;eved ;n one step by react;on of the glass beads w;th concentrated aqueous ammonia (3 ml) at 50C
for 16 hours. The glass beads are then thoroughly washed - with 50X aqueous methanol (3 times with 3 ml each ~ime).
The liquid phase is removed by evaporat;on (removal of the methanol) and freeze-dry;ng. Then an al;quot is filtered through a millipore filter and purified by HPLC on RP 18 as can be seen in Figure 3.
The fractions which co~ntain the 5 -dimethoxy-tritylated oligonucleotide are collected; the volatile buffer is removed in a rotary evaporator in vacuo. 1 ml of 80X strength acet;c ac;d is added to the res;due.
After 45 minutes at room temperature, the acetic acid is removed by freeze-dry;ng.
The mater;al thus obta;ned ;s phosphorylated ;n the customary manner (L;eb;gs Ann. Chem. 19?8, 982) w;th ~Z~406~
T4-polynucleotide kinase and ~-32P-ATP. The resulting product is characterized by polyacrylamide gel electro-phoresis comparing with a homo-oligo-dT chain length standard tNucleic Acids Res. 6, 2096 (1979), Figure 4) and by sequencing accord;ng to Figure 5 tLiebigs Ann.
Chem. 1978, 982).
Figures 6a to 6c show the results (HPLC, gel electrophoresis, sequencing) of the synthesis of d(GGGATCCC) using the nucleoside ~-cyanoethyl N,N-dimethylphosphor-amid;tes. Figures 6a to 6c show the results ~HPLC, 9,electrophoresis, sequencing) of the synthesis of d(GGG
ATATCCC) using the nucleoside ~-cyanoethyl N,N-morpho-linophosphoramidites.
The results given ;n Figures 3, 6a and 7a were ob-tained by using a gradient from 10 to 25 vol. % CH3CN,5 min, and 25 to 29 vol. X CH3CN, 30 min, in 0.1 M tri-ethylammonium acetate at pH 7Ø

lZ34061~) ,~ ~
o ~ ~ N `J
O ~t'`J N +
O CL
C ~ N N C_' -- ~ N ~ ~ N
O N I -- I N ~
~ Q E `~ N 00 C- Q ~ O Q ~ N 11') ~ O Q ~ N
t~ E N ~ E ~ N ~
Z N ~ 11 ^--' _ ~ ~ ~ + _ ~
~) ~ X

I ^ ~ a~ Q Vl ~ N I O _ol 0 E ~ N (~J ~ ~
~ O ~ S o~
Q O I ~ N ~ O Q
~ E ~ N I (_~ ~Vl O E ' ~ ~ ~ ~' 0 00 -- ~ I~ I ~ ~ Z C ~, ~ O E ~ I
Q N ~ I Z ~ `O ICL
.~, I O Q ~. Z~ O ~ ~
O N ~ N ~ It~ V) z 1 0 N 1~- ` + -- ~ C
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Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of oligonucleotides of the general formula I

(I) in which B denotes a nucleobase, R1 denotes hydrogen, hyd-roxyl or hydroxyl which is protected by the protective groups customary in nucleotide chemistry, and r denotes an integer from 1 to 200, comprising the steps of:
a) reacting nucleoside of the general formula II

(II) in which R1 is defined as above, and R2 denotes a pro-tective group customary in nucleotide chemistry and B' de-notes the nucleobase B protected, where appropriate, by the protective groups customary in nucleotide chemistry, with a phosphine derivative of the general formula III
(III) in which R3 is a protective group which can be eliminated, and X
and L are groups which react with hydroxyl groups in the sugar moieties of the nucleotides of nucleosides, in the presence of a base to thereby form a nucleotide derivative as further defined below, b) reacting the nucleotide derivative of the formula IV obtained in step a) and represented by the formula IV:

(IV) in which B', R1, R2, R3 and L are as defined above, with a nucle-oside, of the general formula V, bound to a polymeric carrier (V) in which B' and Rl are as defined above and C denotes the poly-meric carrier c) oxidizing the carrier-bound nucleoside-nucleotide of the gen-eral formula VI, obtained in step b) and represented by the for-mula:
(VI) in which B', R1, R2, R3 and C are as defined above, with forma-tion of phosphotriester groups, d) blocking free primary 5'-OH groups, which have not been re-acted in the reaction according to step b), with permanent pro-tective groups customary in nucleotide chemistry;
e) eliminating the protective group R2;
f) optionally repeating steps a) to e) to introduce further nucleoside phosphate or oligonucleoside phosphate units; and g) cleaving the nucleoside-carrier bond and when required elimi-nating protective groups present in the oliogonucleoside phos-phates which process comprises using the step a) as the phosphine derivative of the general formula III a compound in which R3 de-notes a group of the formula VII

(VII) in which the groups Y, which can be identical or different, represent hydrogen, methyl and/or ethyl and Z represents an electron-attracting group, where, in the phosphine derivative of the formula III, X is chlorine, bromine, CN or SCN and L
is CN or SCN, a secondary amino radical of the formula (VIII) - NR? (VIII) where the groups R4 are primary, secondary or tertiary alkyl radicals having 1 - 10 carbon atoms, or together form a cycloalkyl radical having 5 - 7 carbon atoms, which can con-tain one or two nitrogen, oxygen or sulfur atoms as hetero-atoms, or are imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole, optionally sub-stituted in the phenyl moiety, or benzohydroxytriazole, op-tionally substituted in the phenyl moiety.
2. The process as claimed in claim 1, in which is used a phosphine derivative of the formula III in which X is chlorine or bromine, and L is a secondary amino radical of the formula (VIII) - NR?
(VIII) where the groups R4 are primary, secondary or tertiary alkyl radicals having 1 - 10 carbon atoms, or together form a cycloalkyl radical having 5 - 7 carbon atoms, which can con-tain one or two nitrogen, oxygen or sulfur atoms as hetero-atoms, or are imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole, optionally substituted in the phenyl moiety, or benzohydroxytriazole, optionally substituted in the phenyl moiety.
3. The process as claimed in claim 1 or 2, in which is used a phosphine derivative of the formula (III) in which X is chlorine, L is an N,N-dimethylamino, -diethylamino or -diisopropylamino group or N-*norpholino group, and R3 is a cyanoethyl group.
4. A protected nucleotide having the formula:

where, B1 is a protected nucleobase; R1 is H, OH, or a hydroxyl group which is protected by a protective group customary in nucleotide chemistry; R2 is a protective group;

L is CN, SCN, or NH?; R4 is a primary, secondary or tertiary alkyl radical having 1 - 10 carbon atoms, or where R? is a cycloalkyl radical having 5 - 7 carbon atoms which may contain one or two nitrogen, oxygen or sulfur atoms as heteroatoms; Y is H, CH3, or CH2CH3; and Z is a halogen, CN, NO2, phenylthio, phenylsulfoxy, or phenylsulfonyl, where the phenyl radicals, may be substituted in the o, o, or p positions with a halogen, CN, or NO2 radical, or where the group may be replaced by CF3, CCl3, or CBr3.
5. A protected nucleotide as in claim 4, wherein Z is CN.
6. A protected nucleotide as in claim 5, wherein R3 is CH2-CH2-CN.
7. A protected nucleotide as in claim 4, wherein R2 is 4,4'-dimethoxytrityl or 4,4''-trimethoxytrityl.
8. A protected nucleotide having the formula:

where, R1 is H, OH, or a hydroxyl group which is protected by a protective group customary in nucleotide chemistry; R2 is 4,4'dimethoxytrityl or 4,4',4"-trimethoxytrityl, B' is a pro-tected nucleobase; R3 is CH2-CH2-CN; and L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, or N-morpholino
9. A method of preparing oligonucleotides of the general formula:

wherein B is a nucleobase, R1 is hydrogen, hydroxyl or hydroxyl which is protected by nucleoside protective groups, and n denotes an integer from 1 to 200, comprising the steps of:
a) reacting a nucleotide phosphite represented by the formula:

wherein B' is a protected nucleobase B, R1 is as defined above, R2 is 4,4' dimeth-oxytrityl or 4,4',4'' trimethoxytrityl; and L is N,N-dimethylamino, N,N-diethyl-amino, N, N-diisopropylamino, or N-morpholino, with a nucleoside bound to a polymeric carrier, of the general formula:

wherein B' and R1 are as defined above and C
represents the polymeric carrier to produce a carrier bound nucleoside-nucleotide of the formula:

wherein B', R1, R2, R3 and C are as defined above, b) oxidizing the carrier bound nucleo-side-nucleotide;

c) blocking free primary 5'-OH groups, which have not been re-acted in the reaction of step a), with permanent protective groups;
d) eliminating the protective group R2;
f) repeating steps a) to d) to introduce further nucleoside phos-phate or oligonucleoside phosphate units; and g) cleaving the nucleoside-carrier bond and when required elimi-nating protective groups present in the oligonucleoside phos-phates.
10. A method of synthesizing oligonucleotides, compris-ing the steps of:
a) coupling a nucleoside .beta.-cyanoethyl-protected phosphoramidite to a carrier-bound nucleoside to produce a carrier bound nucleo-side-nucleotide having a phosphite triester linkage;
b) oxidizing the phosphite triester form a phosphate triester;
c) when required coupling additional nucleoside .beta.-cyanoethyl -protected phosphoramidites to the carrier bound nucleoside-nucleotide and after each coupling step, oxidizing the resulting phosphite triester to form a phosphate triester, to form a car-rier bound polynucleotide;
d) removing the .beta.-cyanoethyl protecting groups; and e) removing the polynucleotide from the carrier.
11. A method of claim 10, wherein the nucleoside .beta.-cyanoethyl phosphoramidite is a nucleoside .beta.-cyanoethyl N,N-dimethylphosphoramidite, N,N-diethylphosphoramidite, N,N-dipropyl-phosphoramidite or N,N-morpholino phos-phoramidite.
12. A method of claim 11, wherein the carrier is controlled pore glass.
13. A method of claim 12, wherein the .beta.-cyanoethyl protecting group is removed with simultaneous removal of the polynucleotide from the carrier, by concentrated aqueous ammonia.
14. In a method of polynucleotide synthesis, com-prising sequentially coupling nucleotide phos-phoramidites to produce a polynucleotide, wherein the phosphorus atoms of the nucleotide phosphor-amidites are protected by methyl groups, the improvement wherein the phosphorus protecting group are cyanoethyl groups.
CA000461270A 1983-08-18 1984-08-17 Process for the preparation of oligonucleotides Expired CA1234060A (en)

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Families Citing this family (389)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4973679A (en) * 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US5166330A (en) * 1983-09-06 1992-11-24 Hoechst Aktiengesellschaft Process for the preparation of nucleoside alkyl-aralkyl- and aryl-phosphonites and -phosphonates
EP0222889B1 (en) * 1985-05-15 1993-10-27 Amoco Corporation Cytidine analogs
EP0216357A3 (en) * 1985-09-25 1988-08-31 Nippon Zeon Co., Ltd. Phosphoramidite compounds and process for production thereof
US4959463A (en) * 1985-10-15 1990-09-25 Genentech, Inc. Intermediates
DE3788914T2 (en) * 1986-09-08 1994-08-25 Ajinomoto Kk Compounds for cleaving RNA at a specific position, oligomers used in the preparation of these compounds and starting materials for the synthesis of these oligomers.
WO1988003149A1 (en) * 1986-10-30 1988-05-05 Daicel Chemical Industries, Ltd. Process for synthesizing oligonucleotides and compounds for forming high-molecular protective group
US4835104A (en) * 1987-06-16 1989-05-30 Ajinomoto Co., Inc., Patent & Licensing Department Process for producing and purifying 2',3'-dideoxynucleosides, and process for producing 2',3'-dideoxy-2',3'-didehydronucleosides
US6306647B1 (en) 1987-06-16 2001-10-23 Ajinomoto Co., Inc. Process for producing and purifying 2′,3′-dideoxynucleosides, and process for producing 2′,3′-dideoxy-2′,3′-didehydronucleosides
US5585481A (en) * 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US6031091A (en) * 1987-09-21 2000-02-29 Gen-Probe Incorporated Non-nucleotide linking reagents for nucleotide probes
EP0406309A4 (en) * 1988-03-25 1992-08-19 The University Of Virginia Alumni Patents Foundation Oligonucleotide n-alkylphosphoramidates
WO1989009780A1 (en) * 1988-04-15 1989-10-19 Chemical Industry Institute Of Toxicology Site-specific tritium-labeled oligodeoxynucleotides
US5750666A (en) * 1988-05-26 1998-05-12 Competitve Technologies, Inc. Polynucleotide phosphorodithioate compounds
US5602244A (en) * 1988-05-26 1997-02-11 Competitive Technologies, Inc. Polynucleotide phosphorodithioate compounds
JP3028814B2 (en) * 1988-06-28 2000-04-04 ベイラー カレッジ オブ メディシン Porous wafers for split synthesis of biopolymers
JPH0291088A (en) * 1988-09-29 1990-03-30 Central Glass Co Ltd Phosphite and nucleoside-3'-phosphite derivative and synthesis of oligonucleotide using the same
CA2000805C (en) * 1988-10-17 1994-01-18 Kiyoshi Sudani Carbon/metal composite
US5047524A (en) * 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5262530A (en) * 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
DE3916871A1 (en) * 1989-05-24 1990-11-29 Boehringer Mannheim Gmbh MODIFIED PHOSPHORAMIDITE PROCESS FOR THE PREPARATION OF MODIFIED NUCLEIC ACIDS
US7049102B1 (en) 1989-09-22 2006-05-23 Board Of Trustees Of Leland Stanford University Multi-gene expression profile
US5545522A (en) 1989-09-22 1996-08-13 Van Gelder; Russell N. Process for amplifying a target polynucleotide sequence using a single primer-promoter complex
US5914396A (en) * 1990-01-11 1999-06-22 Isis Pharmaceuticals, Inc. 2'-O-modified nucleosides and phosphoramidites
US5646265A (en) * 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5151510A (en) * 1990-04-20 1992-09-29 Applied Biosystems, Inc. Method of synethesizing sulfurized oligonucleotide analogs
CA2094803A1 (en) * 1990-10-26 1992-04-27 Robert E. Klem Process for the synthesis of oligomers
US6335434B1 (en) 1998-06-16 2002-01-01 Isis Pharmaceuticals, Inc., Nucleosidic and non-nucleosidic folate conjugates
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US8153602B1 (en) 1991-11-19 2012-04-10 Isis Pharmaceuticals, Inc. Composition and methods for the pulmonary delivery of nucleic acids
AU3222793A (en) * 1991-11-26 1993-06-28 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
TW393513B (en) * 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US6235887B1 (en) 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines
ZA929351B (en) * 1991-12-11 1993-06-04 Igen Inc Electrochemiluminescent label for DNA assays.
EP0616612A4 (en) 1991-12-12 1995-01-11 Gilead Sciences Inc Nuclease stable and binding competent oligomers and methods for their use.
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
CA2153387A1 (en) 1993-01-07 1994-07-21 Hubert Koester Dna sequencing by mass spectrometry
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5955591A (en) * 1993-05-12 1999-09-21 Imbach; Jean-Louis Phosphotriester oligonucleotides, amidites and method of preparation
US5428149A (en) * 1993-06-14 1995-06-27 Washington State University Research Foundation Method for palladium catalyzed carbon-carbon coulping and products
KR960703170A (en) * 1993-06-23 1996-06-19 알버트 디. 프리센. ANTISENSE OLIGONUCLEOTIDES AND THERAPEUTIC USE THEREOF IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION
US5571902A (en) * 1993-07-29 1996-11-05 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US5614621A (en) * 1993-07-29 1997-03-25 Isis Pharmaceuticals, Inc. Process for preparing oligonucleotides using silyl-containing diamino phosphorous reagents
US6001982A (en) * 1993-07-29 1999-12-14 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
US6294664B1 (en) 1993-07-29 2001-09-25 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides
EP0728139B1 (en) 1993-09-03 2003-08-13 Isis Pharmaceuticals, Inc. Amine-derivatized nucleosides and oligonucleosides
US6639061B1 (en) 1999-07-07 2003-10-28 Isis Pharmaceuticals, Inc. C3′-methylene hydrogen phosphonate oligomers and related compounds
US5510476A (en) * 1994-07-07 1996-04-23 Isis Pharmaceuticals, Inc. Carbocation scavenging during oligonucleotide synthesis
US6558633B1 (en) * 1994-09-21 2003-05-06 Isis Pharmaceuticals, Inc. Chemical reaction apparatus and methods
USRE43097E1 (en) 1994-10-13 2012-01-10 Illumina, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US6428955B1 (en) 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US20060063193A1 (en) * 1995-04-11 2006-03-23 Dong-Jing Fu Solid phase sequencing of double-stranded nucleic acids
US7803529B1 (en) * 1995-04-11 2010-09-28 Sequenom, Inc. Solid phase sequencing of biopolymers
US5750341A (en) 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5830655A (en) 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US6111095A (en) * 1995-06-07 2000-08-29 Merck & Co., Inc. Capped synthetic RNA, analogs, and aptamers
DE69637285T2 (en) 1995-06-07 2008-07-10 Solexa, Inc., Hayward Oligonucleotide tags for sorting and identification
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
EP0863910A1 (en) 1995-10-19 1998-09-16 NeXstar Pharmaceuticals, Inc. Method for solution phase synthesis of oligonucleotides
US6160109A (en) * 1995-10-20 2000-12-12 Isis Pharmaceuticals, Inc. Preparation of phosphorothioate and boranophosphate oligomers
US6476216B1 (en) 1995-10-20 2002-11-05 Mcgill University Preparation of phosphorothioate oligomers
US5734041A (en) 1995-10-20 1998-03-31 Mcgill University Preparation of chiral phosphorothioate oligomers
US5705621A (en) * 1995-11-17 1998-01-06 Isis Pharmaceuticals, Inc. Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same
US20030119724A1 (en) * 1995-11-22 2003-06-26 Ts`O Paul O.P. Ligands to enhance cellular uptake of biomolecules
US5808035A (en) * 1995-12-08 1998-09-15 Usher; David A. Protected nucleoside and method for its synthesis
WO1997036005A1 (en) * 1996-03-26 1997-10-02 Lynx Therapeutics, Inc. Oligonucleotide treatments and compositions for human melanoma
US5959100A (en) * 1996-03-27 1999-09-28 Nexstar Pharmaceuticals, Inc. Pyrimidine nucleosides as therapeutic and diagnostic agents
US20040147022A1 (en) * 1996-06-06 2004-07-29 Baker Brenda F. 2'-methoxy substituted oligomeric compounds and compositions for use in gene modulations
US20040203024A1 (en) * 1996-06-06 2004-10-14 Baker Brenda F. Modified oligonucleotides for use in RNA interference
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US20040171031A1 (en) * 1996-06-06 2004-09-02 Baker Brenda F. Sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US20050119470A1 (en) * 1996-06-06 2005-06-02 Muthiah Manoharan Conjugated oligomeric compounds and their use in gene modulation
US20070275921A1 (en) * 1996-06-06 2007-11-29 Isis Pharmaceuticals, Inc. Oligomeric Compounds That Facilitate Risc Loading
US20050053976A1 (en) * 1996-06-06 2005-03-10 Baker Brenda F. Chimeric oligomeric compounds and their use in gene modulation
US7812149B2 (en) * 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US5777324A (en) 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US7285422B1 (en) * 1997-01-23 2007-10-23 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US6024925A (en) 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
US6133436A (en) * 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
EP1164203B1 (en) 1996-11-06 2007-10-10 Sequenom, Inc. DNA Diagnostics based on mass spectrometry
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
DE69727489T2 (en) 1996-11-06 2004-11-25 Sequenom, Inc., San Diego METHOD OF MASS SPECTROMETRY
US6060240A (en) * 1996-12-13 2000-05-09 Arcaris, Inc. Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom
US6172217B1 (en) 1996-12-27 2001-01-09 Isis Pharmaceuticals Inc. Method of synthesizing phosphorothioate oligonucleotides
US5902881A (en) * 1997-03-03 1999-05-11 Isis Pharmaceuticals, Inc. Reagent useful for synthesizing sulfurized oligonucleotide analogs
US5760209A (en) * 1997-03-03 1998-06-02 Isis Pharmaceuticals, Inc. Protecting group for synthesizing oligonucleotide analogs
US6207370B1 (en) 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides
US6268131B1 (en) 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US7427678B2 (en) * 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US6326479B1 (en) 1998-01-27 2001-12-04 Boston Probes, Inc. Synthetic polymers and methods, kits or compositions for modulating the solubility of same
US6020475A (en) * 1998-02-10 2000-02-01 Isis Pharmeuticals, Inc. Process for the synthesis of oligomeric compounds
US7273933B1 (en) 1998-02-26 2007-09-25 Isis Pharmaceuticals, Inc. Methods for synthesis of oligonucleotides
US6531590B1 (en) * 1998-04-24 2003-03-11 Isis Pharmaceuticals, Inc. Processes for the synthesis of oligonucleotide compounds
US6780591B2 (en) * 1998-05-01 2004-08-24 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7875440B2 (en) 1998-05-01 2011-01-25 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US6723564B2 (en) 1998-05-07 2004-04-20 Sequenom, Inc. IR MALDI mass spectrometry of nucleic acids using liquid matrices
US6274725B1 (en) 1998-06-02 2001-08-14 Isis Pharmaceuticals, Inc. Activators for oligonucleotide synthesis
US6335439B1 (en) 1998-06-11 2002-01-01 Isis Pharmaceuticals, Inc. Method of preparing phosphoramidites
US6919437B1 (en) 1998-06-11 2005-07-19 Isis Pharmaceuticals, Inc. Synthetic methods and intermediates for triester oligonucleotides
US6326478B1 (en) 1998-07-08 2001-12-04 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US6867294B1 (en) 1998-07-14 2005-03-15 Isis Pharmaceuticals, Inc. Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages
US6242589B1 (en) 1998-07-14 2001-06-05 Isis Pharmaceuticals, Inc. Phosphorothioate oligonucleotides having modified internucleoside linkages
US6124484A (en) 1998-07-17 2000-09-26 Isis Pharmaceuticals, Inc. Recovery of triarylmethyl halide protecting groups cleaved during oligonucleotide synthesis
US6043352A (en) 1998-08-07 2000-03-28 Isis Pharmaceuticals, Inc. 2'-O-Dimethylaminoethyloxyethyl-modified oligonucleotides
US6673912B1 (en) 1998-08-07 2004-01-06 Isis Pharmaceuticals, Inc. 2′-O-aminoethyloxyethyl-modified oligonucleotides
US20040009938A1 (en) * 1998-08-07 2004-01-15 Muthiah Manoharan Methods of enhancing renal uptake of oligonucleotides
US6335437B1 (en) 1998-09-07 2002-01-01 Isis Pharmaceuticals, Inc. Methods for the preparation of conjugated oligomers
US6069243A (en) * 1998-10-06 2000-05-30 Isis Pharmaceuticals, Inc. Process for oligonucleotide synthesis
US6153411A (en) * 1998-10-30 2000-11-28 American Water Works Company, Inc. Methods and kits for detection of Cryptosporidium parvum using immunomagnetic separation and amplification
US6169177B1 (en) 1998-11-06 2001-01-02 Isis Pharmaceuticals, Inc. Processes for the synthesis of oligomeric compounds
US6264891B1 (en) * 1998-12-22 2001-07-24 Eos Biotechnology, Inc. Apparatus and method for concurrent chemical synthesis
US6380378B1 (en) 1998-12-24 2002-04-30 Toagosei Company, Ltd. Nucleotide compound, nucleotide block oligonucleotide, and method for producing them
US6465628B1 (en) * 1999-02-04 2002-10-15 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US6207819B1 (en) 1999-02-12 2001-03-27 Isis Pharmaceuticals, Inc. Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds
US6225061B1 (en) 1999-03-10 2001-05-01 Sequenom, Inc. Systems and methods for performing reactions in an unsealed environment
US6121437A (en) 1999-03-16 2000-09-19 Isis Pharmaceuticals, Inc. Phosphate and thiophosphate protecting groups
US20040082774A1 (en) * 1999-03-16 2004-04-29 Guzaev Andrei P. Novel phosphate and thiophosphate protecting groups
US6399765B1 (en) * 1999-03-17 2002-06-04 Isis Pharmaceuticals, Inc. Methods for removing dimethoxytrityl groups from oligonucleotides
US6965041B1 (en) 1999-03-24 2005-11-15 The United States Of America As Represented By The Department Of Health And Human Services N-acylphosphoramidites and their use in oligonucleotide synthesis
US6221635B1 (en) 1999-05-06 2001-04-24 The Wistar Institute Methods for solid-phase amplification of DNA template (SPADT) using multiarrays
US6610842B1 (en) 1999-05-06 2003-08-26 Isis Pharmaceuticals, Inc. Processes for the synthesis of oligomers using phosphoramidite compositions
US20070059752A1 (en) * 1999-06-09 2007-03-15 Biosearch Technologies, Inc. Fluorescence energy transfer probes with stabilized conformations
US6818395B1 (en) * 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US7501245B2 (en) * 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US6414135B1 (en) 1999-07-07 2002-07-02 Isis Pharmaceuticals, Inc. C3′-methylene hydrogen phosphonate monomers and related compounds
US6593466B1 (en) 1999-07-07 2003-07-15 Isis Pharmaceuticals, Inc. Guanidinium functionalized nucleotides and precursors thereof
US6147200A (en) * 1999-08-19 2000-11-14 Isis Pharmaceuticals, Inc. 2'-O-acetamido modified monomers and oligomers
US6114518A (en) * 1999-09-30 2000-09-05 Becton, Dickinson And Company Synthesis and use of labelled phosphoramidite compositions
US7019129B1 (en) 2000-05-09 2006-03-28 Biosearch Technologies, Inc. Dark quenchers for donor-acceptor energy transfer
US6936702B2 (en) 2000-06-07 2005-08-30 Li-Cor, Inc. Charge-switch nucleotides
WO2001094609A1 (en) 2000-06-07 2001-12-13 Li-Cor, Inc. Charge-switch nucleotides
EP1176151B1 (en) * 2000-07-28 2014-08-20 Agilent Technologies, Inc. Synthesis of polynucleotides using combined oxidation/deprotection chemistry
DE60138916D1 (en) 2000-09-26 2009-07-16 Boston Probes Inc Probes, probes, methods and kits for detection, identification and / or counting of bacteria
US6693187B1 (en) * 2000-10-17 2004-02-17 Lievre Cornu Llc Phosphinoamidite carboxlates and analogs thereof in the synthesis of oligonucleotides having reduced internucleotide charge
US20020142483A1 (en) 2000-10-30 2002-10-03 Sequenom, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
JP2004536027A (en) 2000-12-01 2004-12-02 ジョーンズ・ホプキンス・ユニーバーシティー Glycosylated / galactosylated peptide conjugates, bifunctional linkers, and nucleotide monomers / polymers, and related compositions and methods of use
US6794141B2 (en) 2000-12-22 2004-09-21 Arcturus Bioscience, Inc. Nucleic acid amplification
CA2440754A1 (en) * 2001-03-12 2002-09-19 Stephen Quake Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US6825338B2 (en) 2001-03-30 2004-11-30 Isis Pharmaceuticals, Inc. Labeled oligonucleotides, methods for making same, and compounds useful therefor
WO2002095356A2 (en) * 2001-05-21 2002-11-28 Aclara Biosciences, Inc. Methods and compositions for analyzing proteins
US7118907B2 (en) * 2001-06-06 2006-10-10 Li-Cor, Inc. Single molecule detection systems and methods
EP1412749A4 (en) * 2001-06-28 2006-12-13 Ia Inc Fiber-optic sensor array
WO2003092581A2 (en) 2001-07-16 2003-11-13 Hk Pharmaceuticals, Inc. Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US6822088B2 (en) 2001-07-17 2004-11-23 Isis Pharmaceuticals, Inc. Synthesis of oligonucleotides on solid support
WO2003048179A2 (en) * 2001-12-03 2003-06-12 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Thermolabile hydroxyl protecting groups and methods of use
US7255874B1 (en) 2001-12-21 2007-08-14 Closure Medical Corporation Biocompatible polymers and adhesives: compositions, methods of making and uses related thereto
GB0200526D0 (en) * 2002-01-11 2002-02-27 Science And Technology The Methods
WO2003062452A2 (en) * 2002-01-23 2003-07-31 Proligo, Llc Methods for the integrated synthesis and purification of oligonucleotides
JP2005518451A (en) * 2002-02-22 2005-06-23 ハネウェル・インターナショナル・インコーポレーテッド Process for producing phosphitylated compounds
DK1502102T3 (en) * 2002-03-11 2009-05-04 Caprotec Bioanalytics Gmbh Compounds and Methods for Analyzing the Proteome
AU2003220249A1 (en) * 2002-03-15 2003-09-29 Arcturus Bioscience, Inc. Improved nucleic acid amplification
US7169916B2 (en) * 2002-04-01 2007-01-30 Isis Pharmaceuticals, Inc. Chloral-free DCA in oligonucleotide synthesis
DE60328674D1 (en) 2002-04-17 2009-09-17 Bioren Inc UNIVERSAL BANKS FOR IMMUNOGLOBULINE
AU2003249681A1 (en) * 2002-05-31 2003-12-19 Diversa Corporation Multiplexed systems for nucleic acid sequencing
US6967247B2 (en) 2002-07-24 2005-11-22 Isis Pharmaceuticals, Inc. Deprotection of phosphorus in oligonucleotide synthesis
WO2004022578A2 (en) * 2002-09-08 2004-03-18 Applera Corporation Methods, compositions and libraries pertaining pna dimer and pna oligomer synthesis
CA2504929C (en) * 2002-11-05 2014-07-22 Charles Allerson Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US9150606B2 (en) * 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US7615629B2 (en) * 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support
WO2004064972A2 (en) * 2003-01-16 2004-08-05 Hk Pharmaceuticals, Inc. Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US7002006B2 (en) * 2003-02-12 2006-02-21 Isis Pharmaceuticals, Inc. Protection of nucleosides
US7745116B2 (en) * 2003-04-08 2010-06-29 Pacific Biosciences Of California, Inc. Composition and method for nucleic acid sequencing
CA2540692C (en) * 2003-06-02 2013-05-28 Isis Pharmaceuticals, Inc. Oligonucleotide synthesis with alternative solvents
US20050170367A1 (en) * 2003-06-10 2005-08-04 Quake Stephen R. Fluorescently labeled nucleoside triphosphates and analogs thereof for sequencing nucleic acids
EP1636342A4 (en) * 2003-06-20 2008-10-08 Isis Pharmaceuticals Inc Oligomeric compounds for use in gene modulation
US20050053981A1 (en) * 2003-09-09 2005-03-10 Swayze Eric E. Gapped oligomeric compounds having linked bicyclic sugar moieties at the termini
WO2005027962A1 (en) 2003-09-18 2005-03-31 Isis Pharmaceuticals, Inc. 4’-thionucleosides and oligomeric compounds
US7125945B2 (en) * 2003-09-19 2006-10-24 Varian, Inc. Functionalized polymer for oligonucleotide purification
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
DK2708541T3 (en) 2003-11-13 2015-02-23 Isis Pharmaceuticals Inc 5,6-dihydroxy-isoindole derivatives as linkers for oligomeric solid phase synthesis
WO2005054441A2 (en) * 2003-12-01 2005-06-16 California Institute Of Technology Device for immobilizing chemical and biomedical species and methods of using same
US7691316B2 (en) * 2004-02-12 2010-04-06 Chemistry & Technology For Genes, Inc. Devices and methods for the synthesis of nucleic acids
EP2248911A1 (en) 2004-02-19 2010-11-10 Helicos Biosciences Corporation Methods and kits for analyzing polynucleotide sequences
US20060046258A1 (en) * 2004-02-27 2006-03-02 Lapidus Stanley N Applications of single molecule sequencing
JP4814084B2 (en) * 2004-03-05 2011-11-09 株式会社キラルジェン Boranophosphate monomer and method for producing oligonucleotide derivative using the same
WO2005085272A1 (en) * 2004-03-05 2005-09-15 Takeshi Wada Boranophosphate monomer and process for producing oligonucleotide derivative therefrom
US8569474B2 (en) * 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US20050239085A1 (en) * 2004-04-23 2005-10-27 Buzby Philip R Methods for nucleic acid sequence determination
US20050260609A1 (en) * 2004-05-24 2005-11-24 Lapidus Stanley N Methods and devices for sequencing nucleic acids
US20070117103A1 (en) * 2005-11-22 2007-05-24 Buzby Philip R Nucleotide analogs
US7476734B2 (en) * 2005-12-06 2009-01-13 Helicos Biosciences Corporation Nucleotide analogs
WO2006007207A2 (en) * 2004-05-25 2006-01-19 Helicos Biosciences Corporation Methods and devices for nucleic acid sequence determination
US20070117104A1 (en) * 2005-11-22 2007-05-24 Buzby Philip R Nucleotide analogs
US20090048192A1 (en) * 2004-06-03 2009-02-19 Isis Pharmaceuticals, Inc. Double Strand Compositions Comprising Differentially Modified Strands for Use in Gene Modulation
US8394947B2 (en) * 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
CA2569419A1 (en) * 2004-06-03 2005-12-22 Isis Pharmaceuticals, Inc. Double strand compositions comprising differentially modified strands for use in gene modulation
US20060024678A1 (en) * 2004-07-28 2006-02-02 Helicos Biosciences Corporation Use of single-stranded nucleic acid binding proteins in sequencing
US7427675B2 (en) * 2004-08-23 2008-09-23 Isis Pharmaceuticals, Inc. Compounds and methods for the characterization of oligonucleotides
US7884086B2 (en) * 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
CA2584799A1 (en) * 2004-10-25 2006-05-04 Operon Holdings, Inc. Amino or thiol linker building block for the synthesis of amino- or thiol-functionalized nucleic acids and methods of making and use thereof
DE602004032245D1 (en) 2004-11-16 2011-05-26 Qiagen Gmbh Gene silencing by hybrid sense DNA and antisense RNA constructs coupled to a peptide for easier uptake into cells
US20060118754A1 (en) * 2004-12-08 2006-06-08 Lapen Daniel C Stabilizing a polyelectrolyte multilayer
WO2006065751A2 (en) * 2004-12-13 2006-06-22 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Cpg oligonucleotide prodrugs, compositions thereof and associated therapeutic methods
US7220549B2 (en) 2004-12-30 2007-05-22 Helicos Biosciences Corporation Stabilizing a nucleic acid for nucleic acid sequencing
US20060172328A1 (en) * 2005-01-05 2006-08-03 Buzby Philip R Methods and compositions for correcting misincorporation in a nucleic acid synthesis reaction
US7482120B2 (en) * 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
US20060263790A1 (en) * 2005-05-20 2006-11-23 Timothy Harris Methods for improving fidelity in a nucleic acid synthesis reaction
US20090270479A1 (en) * 2005-07-12 2009-10-29 Antonio Giordano Genetic and Epigenetic Alterations In the Diagnosis and Treatment of Cancer
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US20070202515A1 (en) * 2005-10-12 2007-08-30 Pathologica, Llc. Promac signature application
US20070117102A1 (en) * 2005-11-22 2007-05-24 Buzby Philip R Nucleotide analogs
US20070128610A1 (en) * 2005-12-02 2007-06-07 Buzby Philip R Sample preparation method and apparatus for nucleic acid sequencing
US7579459B2 (en) * 2005-12-12 2009-08-25 Ngo Nam Q Activator bound solid supports for nucleic acid synthesis via the phosphoramidite approach
US20090305248A1 (en) * 2005-12-15 2009-12-10 Lander Eric G Methods for increasing accuracy of nucleic acid sequencing
US7569686B1 (en) 2006-01-27 2009-08-04 Isis Pharmaceuticals, Inc. Compounds and methods for synthesis of bicyclic nucleic acid analogs
ES2516815T3 (en) 2006-01-27 2014-10-31 Isis Pharmaceuticals, Inc. Analogs of bicyclic nucleic acids modified at position 6
US7397546B2 (en) * 2006-03-08 2008-07-08 Helicos Biosciences Corporation Systems and methods for reducing detected intensity non-uniformity in a laser beam
US20080309926A1 (en) * 2006-03-08 2008-12-18 Aaron Weber Systems and methods for reducing detected intensity non uniformity in a laser beam
AU2007245043A1 (en) * 2006-03-24 2007-11-08 Archemix Corp. Solid support reagents for synthesis
US8586554B2 (en) 2006-05-05 2013-11-19 Isis Pharmaceuticals, Inc. Compounds and methods for modulating expression of PTP1B
DK2066684T3 (en) * 2006-05-11 2012-10-22 Isis Pharmaceuticals Inc 5'-Modified Bicyclic Nucleic Acid Analogs
US7666854B2 (en) * 2006-05-11 2010-02-23 Isis Pharmaceuticals, Inc. Bis-modified bicyclic nucleic acid analogs
WO2008027558A2 (en) 2006-08-31 2008-03-06 Codon Devices, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US7947447B2 (en) 2007-01-16 2011-05-24 Somalogic, Inc. Method for generating aptamers with improved off-rates
AU2008260277C1 (en) 2007-05-30 2014-04-17 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
DK2173760T4 (en) 2007-06-08 2016-02-08 Isis Pharmaceuticals Inc Carbocyclic bicyclic nukleinsyreanaloge
EP2176280B2 (en) * 2007-07-05 2015-06-24 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
US8404830B2 (en) * 2007-07-17 2013-03-26 Somalogic, Inc. Method for generating aptamers with improved off-rates
US8680019B2 (en) * 2007-08-10 2014-03-25 Protelica, Inc. Universal fibronectin Type III binding-domain libraries
US8470966B2 (en) 2007-08-10 2013-06-25 Protelica, Inc. Universal fibronectin type III binding-domain libraries
JP5781762B2 (en) * 2007-08-10 2015-09-24 プロテリックス、インク Universal type III fibronectin binding domain library
US8088904B2 (en) 2007-08-15 2012-01-03 Isis Pharmaceuticals, Inc. Tetrahydropyran nucleic acid analogs
US20090180931A1 (en) 2007-09-17 2009-07-16 Sequenom, Inc. Integrated robotic sample transfer device
WO2009067647A1 (en) * 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
EP2242843B1 (en) * 2007-12-31 2015-05-27 XOMA Technology Ltd. Methods and materials for targeted mutagenesis
US8530640B2 (en) * 2008-02-07 2013-09-10 Isis Pharmaceuticals, Inc. Bicyclic cyclohexitol nucleic acid analogs
WO2009117589A1 (en) 2008-03-21 2009-09-24 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising tricyclic nucleosides and methods for their use
WO2009124238A1 (en) * 2008-04-04 2009-10-08 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising neutrally linked terminal bicyclic nucleosides
US8636978B2 (en) * 2008-06-23 2014-01-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Cyclized NGR peptide compounds, compositions, and methods of their use
EP2346883B1 (en) 2008-09-23 2016-03-23 Scott G. Petersen Self delivering bio-labile phosphate protected pro-oligos for oligonucleotide based therapeutics and mediating rna interference
WO2010036696A1 (en) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Cyclohexenyl nucleic acid analogs
US8501805B2 (en) * 2008-09-24 2013-08-06 Isis Pharmaceuticals, Inc. Substituted alpha-L-bicyclic nucleosides
PL2379084T3 (en) 2008-10-15 2018-04-30 Ionis Pharmaceuticals, Inc. Modulation of factor 11 expression
US20120059045A1 (en) 2008-10-24 2012-03-08 Isis Pharmaceuticals, Inc. Methods of using oligomeric compounds comprising 2'-substituted nucleosides
EP2447274B1 (en) 2008-10-24 2017-10-04 Ionis Pharmaceuticals, Inc. Oligomeric compounds and methods
SG171914A1 (en) 2008-12-02 2011-07-28 Chiralgen Ltd Method for the synthesis of phosphorus atom modified nucleic acids
WO2010065159A1 (en) * 2008-12-02 2010-06-10 Oregon Health & Science University Inhibition of dna polymerases to augment chemotherapeutic and antimicrobial agents
JP2012515534A (en) 2009-01-21 2012-07-12 バーテックス ファーマシューティカルズ インコーポレイテッド Method for amplifying hepatitis C virus nucleic acid
US8536320B2 (en) 2009-02-06 2013-09-17 Isis Pharmaceuticals, Inc. Tetrahydropyran nucleic acid analogs
WO2010101951A1 (en) 2009-03-02 2010-09-10 Alnylam Pharmaceuticals, Inc. Nucleic acid chemical modifications
US7960526B2 (en) * 2009-03-30 2011-06-14 Berry And Associates, Inc. Colorimetric-oxycarbonyl protecting groups for use in organic syntheses
KR101885383B1 (en) 2009-07-06 2018-08-03 웨이브 라이프 사이언시스 리미티드 Novel nucleic acid prodrugs and methods of use thereof
US8927513B2 (en) 2009-07-07 2015-01-06 Alnylam Pharmaceuticals, Inc. 5′ phosphate mimics
US9512164B2 (en) 2009-07-07 2016-12-06 Alnylam Pharmaceuticals, Inc. Oligonucleotide end caps
EP2462153B1 (en) 2009-08-06 2015-07-29 Isis Pharmaceuticals, Inc. Bicyclic cyclohexose nucleic acid analogs
ES2691717T3 (en) 2009-10-30 2018-11-28 Novartis Ag Universal libraries of the binding domain of the inferior side of type III fibronectin type III
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
EP3085791A1 (en) 2009-11-25 2016-10-26 Gen9, Inc. Methods and apparatuses for chip-based dna error reduction
WO2011066185A1 (en) 2009-11-25 2011-06-03 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9217144B2 (en) 2010-01-07 2015-12-22 Gen9, Inc. Assembly of high fidelity polynucleotides
WO2011085102A1 (en) 2010-01-11 2011-07-14 Isis Pharmaceuticals, Inc. Base modified bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2011094580A2 (en) 2010-01-28 2011-08-04 Alnylam Pharmaceuticals, Inc. Chelated copper for use in the preparation of conjugated oligonucleotides
US8716467B2 (en) * 2010-03-03 2014-05-06 Gen9, Inc. Methods and devices for nucleic acid synthesis
WO2011115818A1 (en) 2010-03-17 2011-09-22 Isis Pharmaceuticals, Inc. 5'-substituted bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2011123621A2 (en) 2010-04-01 2011-10-06 Alnylam Pharmaceuticals Inc. 2' and 5' modified monomers and oligonucleotides
CN104069512A (en) 2010-04-12 2014-10-01 私募蛋白质体公司 Aptamers to beta-NGF and their use in treating beta-NGF mediated diseases and disorders
WO2011133871A2 (en) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. 5'-end derivatives
WO2011133876A2 (en) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising acyclic and abasic nucleosides and analogs
US20130260460A1 (en) 2010-04-22 2013-10-03 Isis Pharmaceuticals Inc Conformationally restricted dinucleotide monomers and oligonucleotides
WO2011139695A2 (en) 2010-04-28 2011-11-10 Isis Pharmaceuticals, Inc. Modified 5' diphosphate nucleosides and oligomeric compounds prepared therefrom
EP3173419A1 (en) 2010-04-28 2017-05-31 Ionis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US9127033B2 (en) 2010-04-28 2015-09-08 Isis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom
US20130156845A1 (en) 2010-04-29 2013-06-20 Alnylam Pharmaceuticals, Inc. Lipid formulated single stranded rna
WO2011156278A1 (en) 2010-06-07 2011-12-15 Isis Pharmaceuticals, Inc. Bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2011156202A1 (en) 2010-06-08 2011-12-15 Isis Pharmaceuticals, Inc. Substituted 2 '-amino and 2 '-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom
US20130236968A1 (en) 2010-06-21 2013-09-12 Alnylam Pharmaceuticals, Inc. Multifunctional copolymers for nucleic acid delivery
WO2012016245A2 (en) 2010-07-30 2012-02-02 Novartis Ag Fibronectin cradle molecules and libraries thereof
JP5868324B2 (en) 2010-09-24 2016-02-24 株式会社Ｗａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｊａｐａｎ Asymmetric auxiliary group
WO2012064975A1 (en) 2010-11-12 2012-05-18 Gen9, Inc. Protein arrays and methods of using and making the same
EP3000883B8 (en) 2010-11-12 2018-02-28 Gen9, Inc. Methods and devices for nucleic acids synthesis
CA3077910A1 (en) 2010-11-17 2012-05-24 Ionis Pharmaceuticals, Inc. Modulation of alpha synuclein expression
RU2595873C2 (en) 2010-12-27 2016-08-27 Эли Лилли Энд Компани Bovine viral diarrhoea virus type 1b vaccine compositions and methods for production and use thereof
AR084358A1 (en) 2010-12-27 2013-05-08 Lilly Co Eli COMPOSITIONS AND METHODS FOR IDENTIFYING AND DIFFERING VIRAL COMPONENTS OF MULTIVALENT VACCINES OF THE “BOILING FEVER” (BOVINE RESPIRATORY DISEASE COMPLEX (BRDC))
CA2822958A1 (en) 2010-12-30 2012-07-05 Dow Agrosciences Llc Nucleic acid molecules that confer resistance to coleopteran pests
WO2012092573A2 (en) 2010-12-30 2012-07-05 Dow Agrosciences Llc Nucleic acid molecules that target the vacuolar atpase c subunit and confer resistance to coleopteran pests
BRPI1107329A2 (en) 2010-12-30 2019-11-19 Dow Agrosciences Llc nucleic acid molecules that target the vacuolar atpase h-subunit that confer resistance to Coleoptera pests, plant transformation vector, transformed cell, as well as methods for controlling a Coleoptera pest population, controlling an infestation by said pest, improving the yield of a crop and to produce a transgenic cell
US9012722B2 (en) 2010-12-30 2015-04-21 Dow Agrosciences Llc Nucleic acid molecules that target the RHO1 small GTP-binding protein and confer resistance to coleopteran pests
WO2012170347A1 (en) 2011-06-09 2012-12-13 Isis Pharmaceuticals, Inc. Bicyclic nucleosides and oligomeric compounds prepared therefrom
DK2734208T3 (en) 2011-07-19 2017-06-19 Wave Life Sciences Ltd PROCEDURES FOR SYNTHESIS OF FUNCTIONALIZED NUCLEIC ACIDS
DK3594340T3 (en) 2011-08-26 2021-09-20 Gen9 Inc COMPOSITIONS AND METHODS FOR COLLECTING WITH HIGH ACCURACY OF NUCLEIC ACIDS
DK2756080T3 (en) 2011-09-14 2019-05-20 Translate Bio Ma Inc MULTIMERIC OILONCLEOTID CONNECTIONS
WO2013090457A2 (en) 2011-12-12 2013-06-20 Oncoimmunin Inc. In vivo delivery of oligonucleotides
US9150853B2 (en) 2012-03-21 2015-10-06 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US9221864B2 (en) 2012-04-09 2015-12-29 Isis Pharmaceuticals, Inc. Tricyclic nucleic acid analogs
WO2013154799A1 (en) 2012-04-09 2013-10-17 Isis Pharmaceuticals, Inc. Tricyclic nucleosides and oligomeric compounds prepared therefrom
EP3543350B1 (en) 2012-04-24 2021-11-10 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
ES2683707T3 (en) 2012-05-02 2018-09-27 Ibis Biosciences, Inc. DNA sequencing
EP3438285B1 (en) 2012-05-02 2020-10-14 Ibis Biosciences, Inc. Dna sequencing
CA2873766A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics Inc. Compositions and methods for modulating atp2a2 expression
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
AU2013262700A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating hemoglobin gene family expression
US20150152410A1 (en) 2012-05-16 2015-06-04 Rana Therapeutics, Inc. Compositions and methods for modulating mecp2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
WO2013173645A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating utrn expression
EP2850086B1 (en) 2012-05-18 2023-07-05 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes
IL236303B (en) 2012-06-25 2022-07-01 Gen9 Inc Methods for nucleic acid assembly and high throughput sequencing
CN104661664B (en) 2012-07-13 2020-07-03 波涛生命科学有限公司 Chiral control
KR101850319B1 (en) 2012-07-13 2018-04-20 웨이브 라이프 사이언시스 리미티드 Asymmetric auxiliary group
CN104684923B (en) 2012-07-13 2018-09-28 株式会社新日本科学 Chiral Nuclec acid adjuvants
US9403865B2 (en) 2012-08-15 2016-08-02 Ionis Pharmaceuticals, Inc. Method of preparing oligomeric compounds using modified capping protocols
AU2013306006B2 (en) 2012-08-20 2017-07-06 The Regents Of The University Of California Polynucleotides having bioreversible groups
JP2015529469A (en) 2012-09-14 2015-10-08 ラナ セラピューティクス インコーポレイテッド Multimeric oligonucleotide compounds
US9029335B2 (en) 2012-10-16 2015-05-12 Isis Pharmaceuticals, Inc. Substituted 2′-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2014063051A2 (en) 2012-10-18 2014-04-24 Idexx Laboratories, Inc. Nucleic acid amplification controls and kits and methods of use thereof
WO2014072832A2 (en) 2012-10-18 2014-05-15 Oslo Universitetstssykehus Hf Biomarkers for cervical cancer
WO2014120861A2 (en) 2013-01-31 2014-08-07 Isis Pharmaceuticals, Inc. Method of preparing oligomeric compounds using modified coupling protocols
EP2971034B1 (en) 2013-03-13 2020-12-02 Gen9, Inc. Compositions, methods and apparatus for oligonucleotides synthesis
RU2686080C2 (en) 2013-05-01 2019-04-24 Ионис Фармасьютикалз, Инк. Compositions and methods
CA2921459A1 (en) 2013-08-16 2015-02-19 Rana Therapeutics, Inc. Oligonucleotides targeting euchromatin regions of genes
WO2015023937A1 (en) * 2013-08-16 2015-02-19 Rana Therapeutics, Inc. Heterochromatin forming non-coding rnas
US11162096B2 (en) 2013-10-14 2021-11-02 Ionis Pharmaceuticals, Inc Methods for modulating expression of C9ORF72 antisense transcript
WO2015061246A1 (en) 2013-10-21 2015-04-30 Isis Pharmaceuticals, Inc. Method for solution phase detritylation of oligomeric compounds
RU2732402C2 (en) 2013-11-21 2020-09-16 Сомалоджик, Инк. Cytidine-5-carboxamide modified nucleotide compositions and methods related thereto
BR102014031844A2 (en) 2013-12-20 2015-10-06 Dow Agrosciences Llc RAS and related nucleic acid molecules that confer resistance to Coleoptera and Hemiptera pests
KR20160093728A (en) 2013-12-20 2016-08-08 다우 아그로사이언시즈 엘엘씨 Rnapii-140 nucleic acid molecules that confer resistance to coleopteran pests
JPWO2015108048A1 (en) 2014-01-15 2017-03-23 株式会社新日本科学 Chiral nucleic acid adjuvant and antitumor agent having antitumor activity
EP3095460A4 (en) 2014-01-15 2017-08-23 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
JPWO2015108047A1 (en) 2014-01-15 2017-03-23 株式会社新日本科学 Chiral nucleic acid adjuvant having immunity induction activity and immunity induction activator
SG10201912897UA (en) 2014-01-16 2020-02-27 Wave Life Sciences Ltd Chiral design
US10036019B2 (en) 2014-03-17 2018-07-31 Ionis Pharmaceuticals, Inc. Bicyclic carbocyclic nucleosides and oligomeric compounds prepared therefrom
WO2015143245A1 (en) 2014-03-19 2015-09-24 Isis Pharmaceuticals, Inc. Methods for modulating ataxin 2 expression
SG11201607761UA (en) 2014-03-19 2016-10-28 Ionis Pharmaceuticals Inc Compositions for modulating ataxin 2 expression
PT3757214T (en) 2014-04-01 2022-08-26 Biogen Ma Inc Compositions for modulating sod-1 expression
WO2015164693A1 (en) 2014-04-24 2015-10-29 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising alpha-beta-constrained nucleic acid
EP3137476B1 (en) 2014-04-28 2019-10-09 Ionis Pharmaceuticals, Inc. Linkage modified oligomeric compounds
PE20170010A1 (en) 2014-05-01 2017-03-04 Ionis Pharmaceuticals Inc COMPOSITIONS AND METHODS TO MODULATE THE EXPRESSION OF THE COMPLEMENT FACTOR B
EP3137115B1 (en) 2014-05-01 2020-10-14 Ionis Pharmaceuticals, Inc. Method for synthesis of reactive conjugate clusters
CA2947756A1 (en) 2014-05-07 2015-11-12 Dow Agrosciences Llc Dre4 nucleic acid molecules that confer resistance to coleopteran pests
US10093967B2 (en) 2014-08-12 2018-10-09 The Regents Of The University Of Michigan Detection of nucleic acids
ES2811279T3 (en) 2014-12-22 2021-03-11 Fraunhofer Ges Forschung Nucampholin nucleic acid molecules for controlling beetle insect pests
US9688707B2 (en) 2014-12-30 2017-06-27 Ionis Pharmaceuticals, Inc. Bicyclic morpholino compounds and oligomeric compounds prepared therefrom
WO2016112132A1 (en) 2015-01-06 2016-07-14 Ionis Pharmaceuticals, Inc. Compositions for modulating expression of c9orf72 antisense transcript
WO2016115490A1 (en) 2015-01-16 2016-07-21 Ionis Pharmaceuticals, Inc. Compounds and methods for modulation of dux4
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
WO2016137923A1 (en) 2015-02-23 2016-09-01 Ionis Pharmaceuticals, Inc. Method for solution phase detritylation of oligomeric compounds
US11129844B2 (en) 2015-03-03 2021-09-28 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating MECP2 expression
EP3067424A1 (en) 2015-03-13 2016-09-14 Dow AgroSciences LLC Rna polymerase i1 nucleic acid molecules to control insect pests
WO2016167780A1 (en) 2015-04-16 2016-10-20 Ionis Pharmaceuticals, Inc. Compositions for modulating expression of c9orf72 antisense transcript
BR102016012010A2 (en) 2015-05-29 2020-03-24 Dow Agrosciences Llc NUCLEIC ACID, RIBONUCLEIC ACID (RNA) AND DOUBLE-FILAMENT RIBONUCLEIC ACID (DSRNA) MOLECULE, CELL, PLANT AND SEED USES, PRIMARY PRODUCT, AS WELL AS METHODS TO CONTROL A POPULATION OF HOLIDAYS, OR HOSPITALS, OR HOSPITALS, OR HOSPITALS, OR HOSPITALS THE INCOME OF A CULTURE, AND TO PRODUCE A TRANSGENIC VEGETABLE CELL AND A TRANSGENIC PLANT
MA43072A (en) 2015-07-22 2018-05-30 Wave Life Sciences Ltd COMPOSITIONS OF OLIGONUCLEOTIDES AND RELATED PROCESSES
US10533175B2 (en) 2015-09-25 2020-01-14 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating Ataxin 3 expression
US11058709B1 (en) 2015-12-04 2021-07-13 Ionis Pharmaceuticals, Inc. Methods of treating breast cancer
US10907160B2 (en) 2016-01-05 2021-02-02 Ionis Pharmaceuticals, Inc. Methods for reducing LRRK2 expression
WO2017161172A1 (en) 2016-03-16 2017-09-21 Ionis Pharmaceuticals, Inc. Methods of modulating keap1
WO2017161168A1 (en) 2016-03-16 2017-09-21 Ionis Pharmaceuticals, Inc. Modulation of dyrk1b expression
EP3471781A4 (en) 2016-06-17 2020-05-06 Ionis Pharmaceuticals, Inc. Modulation of gys1 expression
WO2018055577A1 (en) 2016-09-23 2018-03-29 Synthena Ag Mixed tricyclo-dna, 2'-modified rna oligonucleotide compositions and uses thereof
CN109661233A (en) 2016-10-06 2019-04-19 Ionis 制药公司 The method that oligomeric compound is conjugated
JOP20190104A1 (en) 2016-11-10 2019-05-07 Ionis Pharmaceuticals Inc Compounds and methods for reducing atxn3 expression
WO2018098264A1 (en) * 2016-11-23 2018-05-31 Wave Life Sciences Ltd. Compositions and methods for phosphoramidite and oligonucleotide synthesis
EP3548620A4 (en) 2016-12-02 2020-07-22 Cold Spring Harbor Laboratory Modulation of lnc05 expression
EP3342780A1 (en) 2016-12-30 2018-07-04 Dow AgroSciences LLC Pre-mrna processing factor 8 (prp8) nucleic acid molecules to control insect pests
WO2018165564A1 (en) 2017-03-09 2018-09-13 Ionis Pharmaceuticals, Inc. Morpholino modified oligomeric compounds
WO2019006455A1 (en) 2017-06-30 2019-01-03 Solstice Biologics, Ltd. Chiral phosphoramidite auxiliaries and methods of their use
US11197884B2 (en) 2017-08-18 2021-12-14 Ionis Pharmaceuticals, Inc. Modulation of the notch signaling pathway for treatment of respiratory disorders
WO2019051173A1 (en) 2017-09-08 2019-03-14 Ionis Pharmaceuticals, Inc. Modulators of smad7 expression
TWI809004B (en) 2017-11-09 2023-07-21 美商Ionis製藥公司 Compounds and methods for reducing snca expression
WO2019126641A2 (en) 2017-12-21 2019-06-27 Ionis Pharmaceuticals, Inc. Modulation of frataxin expression
MX2020007369A (en) 2018-01-15 2020-10-28 Ionis Pharmaceuticals Inc Modulators of dnm2 expression.
TW202000199A (en) 2018-03-02 2020-01-01 美商Ionis製藥公司 Modulators of IRF4 expression
US11732260B2 (en) 2018-03-02 2023-08-22 Ionis Pharmaceuticals, Inc. Compounds and methods for the modulation of amyloid-β precursor protein
US11661601B2 (en) 2018-03-22 2023-05-30 Ionis Pharmaceuticals, Inc. Methods for modulating FMR1 expression
EP3775204A4 (en) 2018-04-11 2021-12-29 Ionis Pharmaceuticals, Inc. Modulators of ezh2 expression
CN112236438A (en) 2018-04-13 2021-01-15 百时美施贵宝公司 Novel phosphorus (V) -based reagents, method for the production thereof, and use thereof for producing sterically defined organic phosphorus (V) compounds
UY38225A (en) 2018-05-09 2019-11-29 Ionis Pharmaceuticals Inc COMPOUNDS AND METHODS TO REDUCE THE EXPRESSION OF ATXN3
MX2020011913A (en) 2018-05-09 2021-01-29 Ionis Pharmaceuticals Inc Compounds and methods for reducing fxi expression.
WO2019241648A1 (en) 2018-06-14 2019-12-19 Ionis Pharmaceuticals, Inc. Compounds and methods for increasing stmn2 expression
US11332746B1 (en) 2018-06-27 2022-05-17 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing LRRK2 expression
JOP20210018A1 (en) 2018-07-25 2021-01-21 Ionis Pharmaceuticals Inc Compounds and methods for reducing atxn2 expression
TW202028222A (en) 2018-11-14 2020-08-01 美商Ionis製藥公司 Modulators of foxp3 expression
EP3880821A4 (en) 2018-11-15 2023-01-25 Ionis Pharmaceuticals, Inc. Modulators of irf5 expression
JP2022519532A (en) 2019-01-31 2022-03-24 アイオーニス ファーマシューティカルズ， インコーポレーテッド Modulator of YAP1 expression
MX2021010152A (en) 2019-02-27 2021-09-14 Ionis Pharmaceuticals Inc Modulators of malat1 expression.
JP2022527105A (en) 2019-03-29 2022-05-30 アイオーニス ファーマシューティカルズ， インコーポレーテッド Compounds and Methods for Modulating UBE3A-ATS
EP3956450A4 (en) 2019-07-26 2022-11-16 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating gfap
WO2021030778A1 (en) 2019-08-15 2021-02-18 Ionis Pharmaceuticals, Inc. Linkage modified oligomeric compounds and uses thereof
CN111039977B (en) * 2019-12-31 2022-11-08 广州同隽医药科技有限公司 Phosphorylylation reagent and application thereof in oligonucleotide synthesis
TW202140787A (en) 2020-02-28 2021-11-01 美商Ionis製藥公司 Compounds and methods for modulating smn2
WO2021222768A2 (en) 2020-05-01 2021-11-04 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating atxn1
CA3185749A1 (en) 2020-06-29 2022-01-06 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating plp1
EP4136092A4 (en) 2020-11-18 2023-10-11 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating angiotensinogen expression
CA3223192A1 (en) 2021-06-18 2022-12-22 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing ifnar1 expression
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4310662A (en) * 1979-12-26 1982-01-12 Genentech, Inc. Nucleosidic phosphorylating agent and methods
AU550148B2 (en) 1980-02-29 1986-03-06 University Patents Inc. Supports for use in preparation of polynucleotides
US4500707A (en) * 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4458066A (en) * 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
IL62834A0 (en) * 1980-05-14 1981-07-31 Ens Bio Logicals Inc Polynucleotide synthesis
US4415732A (en) * 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
NL8102323A (en) * 1981-05-12 1982-12-01 Unilever Nv PHOSPHORIC ACID DERIVATIVES, METHOD FOR PREPARING THE SAME, AND METHOD FOR PHOSPHORYLATING ORGANIC HYDROXYL COMPOUNDS AND PHOSPHORTRIESTER INTERMEDIARIES FOR USE IN THE SYNTHESIS OF DESOXY AND RIBO-NUCLEIC ACIDS.
US4419509A (en) * 1981-08-24 1983-12-06 Eli Lilly And Company Process for de-cyanoethylating blocked nucleotides
EP0090789A1 (en) * 1982-03-26 1983-10-05 Monsanto Company Chemical DNA synthesis
US4476301A (en) * 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4605735A (en) * 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
NL8302353A (en) * 1983-07-01 1985-02-01 Stichting Katholieke Univ PHOSPHORIZATION AGENT; METHOD FOR PREPARING THEREOF METHOD FOR PHOSPHORYLATING AN ORGANIC HYDROXYL OR AMINE COMPOUND
US4591614A (en) * 1983-10-07 1986-05-27 The Johns Hopkins University Preparation of oligodeoxyribonucleoside alkyl or arylphosphonates

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US4725677A (en) 1988-02-16
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