`

`PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PA TENT COOPERATION TREATY (PCT)
`WO 96/23807
`
`(51) International Patent Classification 6 :
`C07H 19no, 19/20, C12Q V68
`
`Al
`
`(11) International Publleatlon Number:
`
`(43) International Publication Date:
`
`8 August 1996 (08.08.96)
`
`(21) International Appllcation Number:
`
`PCT/SE96/00096
`
`(22) International Filing Date:
`
`30 January 1996 (30.01.96)
`
`(81) Designated States: CA, JP, US, European patent (AT, BE, CH,
`DE, DK, ES, FR, GB, GR, IB, IT, LU, MC, NL, PT, SE).
`
`(30) Priority Data:
`9500342-2
`
`31 January 1995 (31.01.95)
`
`SE
`
`Published
`With international search report.
`
`KWIATKOWSKI, Marek
`(71)(72) Applicant and Inventor:
`[SE/SE]; LOvsAngarvagen 17, S-756 52 Uppsala (SE).
`
`(74) Agents: WID~. BjOm et al.; Pharmacia AB, Patent Dept., S-
`751 82 Uppsala (SE).
`
`(54) Title: NOVEL CHAIN TERMINATORS, THE USE THEREOF FOR NUCLEIC ACID SEQUENCING AND SYNTIIESIS AND A
`METHOD OF THEIR PREPARATION
`
`X
`0
`0
`B
`II
`II
`II
`HO-P-O-P-0-P-07¢1
`I
`I
`I
`OH OH OH
`
`y
`
`0
`I
`R,-z-C-L1-(A) - (L2) 1- (O)m- (F)n
`I
`R2
`
`(I)
`
`(57) Abstract
`
`The invention relates to compounds of general sttucture (I) or salts thereof, wherein B is a nucleobase, X and Z independently are
`oxygen or sulphur, Y is hydrogen or hydroxy, which optionally may be protected, Rt is hydrocarbyl, which optionally is substituted with
`a functional group, R2 is hydrogen or hydrocarbyl, which optionally is substituted with a functional group, A is an electron withdrawing
`or electron donating group capable of moderating the acetal stability of compound (I), Lt and L2 are hydrocarbon linkers, which may be
`the same or different, L2, when present, being either (i) connected to Lt via the group A, or (ii) directly connected to Lt, the group A then
`being connected to one of linkers Lt and L2, Fis a dye label, Q is a coupling group for F, and 1, m and n independently are 0 or I, with
`the proviso that 1 is 1 when m is 1, and 1 is 1 and m is I when n is 1. The compounds of fonnula (I) are useful as deactivatable chain
`extension terminators. The invention also relates to the use of the compounds (I) in nucleic acid synthesis and nucleic acid sequencing as
`well as to a method of preparing compounds of Fonnula (I).
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Annenia
`AM
`AT
`Austria
`Austtalia
`AU
`BB
`Barl>ados
`Belgium
`BE
`Burkina Fuo
`BF
`Bulgaria
`BG
`BJ
`Benin
`BR
`Brazil
`BY
`Belarus
`CA
`Canada
`Central African Republic
`CF
`Congo
`CG
`Switzerland
`CH
`Cl ~ d'Ivoire
`Cameroon
`CM
`CN
`China
`cs
`C:r.«hollovakia
`C:r.«h Republic
`CZ
`DE
`Germany
`DK
`Denmark
`EE
`Estonia
`Spain
`ES
`n
`Finland
`France
`FR
`GA
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`IE
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`u
`LK
`LR
`LT
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`MR
`
`United Kingdom
`Georgia
`Guinea
`Gteece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`XazakhlWl
`Lieclunstein
`Sri Lanka
`Liberia
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`Mauritania
`
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`1T
`UA
`UG
`us
`uz
`VN
`
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New2.ealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`Slovc:nia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`Uganda
`United Stares of America
`Uzbekistan
`Viet Nam
`
`

`

`WO96/23807
`
`1
`
`PCT/SE96/00096
`
`NOVEL CHAIN TERMINATORS, THE USE THEREOF FOR NUCLEIC ACID
`SEQUENCING AND SYNTHESIS AND A METHOD OF THEIR PREPARATION
`
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`The present invention relates to novel nucleic acid
`chain extension terminators, their use in nucleic acid
`sequencing and synthesis, respectively, as well as a
`method for preparing such compounds.
`Today, there are two predominant methods for DNA
`sequence determination: the chemical degradation method
`(Maxam and Gilbert, Proc. Natl. Acad. Sci. ll:560-564
`(1977), and the dideoxy chain termination method (Sanger
`et al., Proc. Natl. Acad. Sci. ll:5463-5467 (1977)). Most
`automated sequencers are based on the chain termination
`method utilizing fluorescent detection of product
`formation. In these systems either primers to which
`deoxynucleotides and dideoxynucleotides are added are qye(cid:173)
`labelled, or the added dideoxynucleotides are
`fluorescently labelled. As an alternative, dye labelled
`deoxynucleotides can be used in conjunction with unlabeled
`dideoxynucleotides. This chain termination method is based
`upon the ability of an enzyme to add specific nucleotides
`onto the 3' hydroxyl end of a primer annealed to a
`template. The base pairing property of nucleic acids
`determines the specificity of nucleotide addition. The
`extension products are then separated electrophoretically
`on a polyacrylamide gel and detected by an optical system
`utilizing laser excitation.
`Although both the chemical degradation method and the
`dideoxy chain termination method are in widespread use,
`there are many associated disadvantages. For example, the
`methods require gel-electrophoretic separation. Typically,
`only 400-800 base pairs can be sequenced from a single
`clone. As a result, the systems are both time- and labor(cid:173)
`intensive. Methods avoiding gel separation have been
`developed in attempts to increase the sequencing
`throughput.
`Sequencing by hybridization (SBH) methods have been
`proposed by Crkvenjakov (Drmanac et al., Genomics i:114
`
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`WO96/l3807
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`PCT/SE96/00096
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`10
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`(1989); Strezoska et al., (Proc. Natl. Acad. Sci. USA
`.B.a:10089 (1991)), Bains and Smith (Bains and Smith, J.
`Theoretical .Biol. llS.:303 (1988)) and in US-A-5,202,231.
`This type of system utilizes the information obtained from
`5 multiple hybridizations of the polynucleotide of interest,
`using short oligonucleotides to determine the nucleic acid
`sequence. These methods potentially can increase the
`sequence throughput beacuse multiple hybridization
`reactions are performed simultaneously. To reconstruct the
`sequence, however, an extensive computer search algorithm
`is required to determine the most likely order of all
`fragments obtained from the multiple hybridizations.
`The SBH methods are problematic in several respects.
`For example, the hybridization is dependent upon the
`sequence composition of the duplex of the oligonucleotide
`and the polynucleotide of interest, so that GC-rich
`regions are more stable than AT-rich regions. As a result,
`false positives and false negatives during hybridization
`detection are frequently present and complicate sequence
`determination. Furthermore, the sequence of the
`polynucleotide is not determined directly, but is inferred
`from the sequence of the known probe, which increases the
`possibility for error.
`Methods have also been proposed which detect the
`addition or removal of single molecules from a DNA strand.
`For example, Hyman E.D., Anal. Biochem., l.lJ.:423
`(1988) discloses the addition of a nucleotide to a an
`immobilised DNA template/primer complex in the presence of
`a polymerase and determination of polymerisation reaction
`by detecting the pyrophosphate liberated as a result of
`the polymerisation.
`Jett et al., J. Biomol. Struct. Dyn., I, p. 301, 1989
`discloses a method wherein a single stranded DNA or RNA
`molecule of labelled nucleotides, complementary to the
`sequence to be determined, is suspended in a moving flow
`stream. Individual bases are then cleaved sequentially
`from the end of the suspended sequence and determined by a
`detector passed by the flow stream.
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`WO96/23807
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`PCT/SE96/00096
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`EP-A-223 618 discloses the use of an immobilised DNA
`template, primer and polymerase exposed to a flow
`containing only one species of deoxynucleotide at a time.
`A downstream detection system then determines whether
`deoxynucleotide is incorporated into the copy or not by
`detecting the difference in deoxynucleotide concentrations
`entering and leaving the flow cell containing the complex
`of DNA template and polymerase.
`WO 90/13666 proposes a method directly measuring the
`growth of the template copy rather than determining it
`indirectly from compositions in the flow medium. Only one
`of the four nucleotides is present at a time, and the
`polymerisation events reflecting the incorporation of a
`nucleotide or not are detected by spectroscopic means
`(evanescent wave spectroscopy, fluorescence detection,
`absorption spectroscopy) or by the individual nucleotides
`being labelled.
`Similar methods employing labelled 3'-blocked
`deoxynucleotides where the blocking group is removable and
`20 which thus permit sequential deoxynucleotide
`addition/detection steps are disclosed in WO 91/06678, US(cid:173)
`A-5,302,509, DE-A-414 1178 and WO 93/21340. However, the
`necessary 3'-blocking groups are either not described in
`any detail, or are not accepted by the required enzyme, or
`do not permit desired rapid deblocking of the growing
`template copy strand after each polymerisation event.
`One object of the present invention is to provide
`novel nucleotide derivatives which may be used as chain
`terminators and which by deprotection may readily be
`converted into nucleotides or nucleotide analogues that
`may be further extended.
`Another object of the present invention is to provide
`a method for nucleotide sequence determination using the
`novel chain terminators.
`Still another object of the present invention is to
`provide a method of synthesizing oligo- or polynucleotides
`by means of the novel chain terminators.
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`WO96/23807
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`PCT/SE96/00096
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`Another object of the present invention is to provide
`a process of preparing novel chain terminators according
`to the invention.
`In accordance with the invention, these objects are
`achieved by the provision of a chain terminating
`nucleotide or nucleotide analogue having its 3'-hydroxyl
`group protected by an acetal or thioacetal structure
`designed in such a way that the 3'-hydroxyl can be
`deprotected in a relatively short time in dilute acid,
`such as hydrochloric acid at pH 2, for example.
`In one aspect, the present invention therefore
`provides a compound of the general formula I:
`
`X
`0
`0
`B
`II
`II
`II
`HO-P-O-P-O-P-O'QO
`I
`I
`I
`OH OH OH
`
`I
`
`V
`
`0
`I
`R1-Z-C-L1-(A)- (L2) 1- (Q)m- (F)n
`I
`R2
`
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`or a salt thereof, such as a trimethylammonium, ammonium,
`sodium or potassium salt, wherein
`Bis a nucleobase,
`X and Z independently are oxygen or sulphur,
`Y is hydrogen, hydroxy or protected hydroxy, such as
`30 methoxy, ethoxy or allyloxy,
`R1 is hydrocarbyl, which optionally is substituted
`with a functional group,
`R2 is hydrogen or hydrocarbyl, which optionally is
`substituted with a functional group,
`A is an electron withdrawing or electron donating
`group capable of moderating the acetal stability of the
`compound I via L1,
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`WO96/23807
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`PCT/SE96/00096
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`L1 and L2 are hydrocarbon linkers, which may be the
`same or different, L2, when present, being either {i}
`connected to L1 via the group A, or (ii) directly
`connected to L1, the group A then being bound to one of
`linkers L1 and L2,
`Fis a dye label,
`Q is a coupling group for F, and
`1, m and n independently are O or 1, with the proviso
`that 1 is 1 when mis 1, and 1 is 1 and mis 1 when n is
`1.
`
`In Formula I above, the nucleobase B may be natural
`or synthetic. Natural nucleobases include common
`nucleobases, such as adenine, guanine, cytosine, thymine
`and uracil, as well as less common nucleobases, such as
`xanthine, hypoxanthine or 2-aminopurine. Synthetic
`nucleobases Bare analogues to the natural nucleobases and
`capable of interacting with other nucleobases in a
`specific, hydrogen bond determined way.
`The hydrocarbyl groups represented by R1 and R2
`include a wide variety, including straight and branched
`chain alkyl, alkenyl, aryl, aralkyl and cycloalkyl,
`preferably containing up to 10 carbon atoms. Preferred
`hydrocarbyl groups are primary, secondary or tertiary
`alkyl, alkenyl or alkynyl groups, especially lower alkyl
`groups, such as methyl and ethyl. The optional functional
`group substituents on R1 and R2 are capable of moderating
`the lability of the 3' acetal group through an inductive
`effect. Exemplary of such functional groups are tert.
`amino, nitro, cyano and halogen (fluoride, chloride,
`bromide, iodide}.
`Detectable moiety, or label F can be chosen from a
`vast number of such moieties known to those skilled in the
`art. Exemplary such moities are radioactively labeled
`functions, luminescent, electroluminescent or fluorescent
`labels, and labels that absorb characteristic visible or
`infrared light. Preferably, Fis a fluorescent label.
`Coupling group Q, when n=O, is a reactive group to
`which a label F can be coupled, or, when n=l, is the
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`WO96/23807
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`PCT/SE96/00096
`
`derivatized residue of a reactive group used for coupling
`linker L2 to label F. A large number of such coupling
`groups capable of reacting with and binding label F via a
`reactive function thereon are known to those skilled in
`the art. Examples of reactive groups are amino, thio, and
`carboxyl. In some cases, the group Q is derived completely
`from the reactive function on the uncoupled F. For
`example, if the coupling reaction is a substitution
`reaction, the group reacting with F being, for example, a
`halogen (fluoride, chloride, bromide or iodide), then the
`group Q will be represented by the reactive function
`present on label F.
`For certain applications of the compounds of Formula
`I, as will be described below, no detectable moiety will
`be needed, and the label F, and optionally also the group
`Q, can then be omitted.
`Electron withdrawing or donating group A is
`incorporated into the structure as a moderator of acetal
`stability. The group A may represent a part of the chain
`L1-A-L2. Representative groups A in that case are amide,
`sulfoxy, sulfone, carbalkoxy (ester), ether, thioether and
`amino groups. Alternatively, A may be a side substituent
`for the L1-L2 chain, representative groups then being, for
`example, cyano, nitre and halogen (halogen including
`fluoride, chloride, bromide and iodide). In this latter
`case, linker L1 will donate structure stretching from the
`acetal carbon to the place of the substitution, and linker
`L2 will donate structure stretching from this substitution
`to the group A. It is to be emphasized that the specific
`electron withdrawing and electron donating groups
`mentioned above are only examples and that many more such
`groups are known and obvious to those skilled in the art.
`The structure of hydrocarbon linker L1 will be
`selected with regard to the function A, inductive effects
`being highly depending on distance. While a straight
`aliphatic (saturated or unsaturated) hydrocarbon chain is
`preferred, branched or cyclic hydrocarbons may be
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`WO96/23807
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`contemplated. Preferably, linker L1 has up to 10 carbon
`atoms, more preferably up to 6 carbon atoms.
`The function of hydrocarbon linker L2 is to provide,
`together with linker L1, A and coupling group Q, for a
`sufficiently long distance between the label moiety F and
`the rest of the compound of Formula I. This is required by
`spatial preferences of the enzymes (polymerases) for which
`the compound of Formula I acts as substrate or by the
`necessity of avoiding interaction between the label and
`nucleobase B. It is readily understood that the structure
`of linker L2 highly depends on the particular enzyme,
`label F and possibly also nucleobase B that are used. A
`suitable length and structure of linker L2 will therefore
`be selected by the skilled person for each particular
`situation. Similarly as for linker L1, a preferred
`structure for linker L2 is a straight aliphatic (saturated
`or unsaturated) hydrocarbon chain, although branched or
`cyclic hydrocarbons may be contemplated. Preferably,
`linker L2 has up to 10 carbon atoms, more preferably up to
`6 carbon atoms.
`In cases where the label F and the coupling group Q
`may be omitted, linker L2 may, of course, also be omitted.
`The compounds of Formula I can be deprotected to
`exhibit a free 3'-hydroxy group in a relatively short time
`under acidic conditions, e.g. with hydrochloric acid at
`about pH 2. It is understood that by proper selection of
`the groups R1, R2, A, L1 and L2 with regard to each other,
`the deprotection time may be adjusted to a desired range.
`Under the mentioned acidic conditions, the most preferred
`compounds of Formula I will be deprotected within, say,
`0.01 to 15 minutes.
`While the compounds of Formula I may be used as pure
`chain extension inhibitors, or chain terminators, for
`example, in DNA sequencing according to the chain
`termination method, as is per se known in the art, the
`advantages of the compounds are, of course, better
`benefited from when the convenient deprotection
`capabilities of the compounds are utilized. This is, for
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`WO96/23807
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`PCT/SE96/00096
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`example, the case when the compounds I are used in nucleic
`acid sequencing methods based on the sequential
`incorporation and determination of individual nucleotides
`in a growing nucleic acid copy strand as described in, for
`example, the aforementioned WO 91/06678, US-A-5,302,509,
`DE-A-414 1178 and WO 93/21340.
`Another aspect of the invention therefore provides a
`method for determining the sequence of a nucleic acid,
`which method comprises providing a single-stranded
`template comprising the nucleic acid to be determined, and
`at least partially synthesizing a complementary nucleic
`acid molecule in a stepwise serial manner by the addition
`of nucleotides in which the identity of each nucleotide
`incorporated into the complementary nucleic acid molecule
`is determined subsequent to its incorporation, wherein
`said nucleotides are compounds of Formula I as defined
`above, and wherein the 3'-blocking group is removed from
`the nucleotide after its incorporation to permit further
`extension of the nucleic acid molecule.
`In one embodiment, a method for determining the
`sequence of a nucleic acid comprises the following steps:
`(i)
`providing a single-stranded template comprising
`the nucleic acid to be sequenced,
`(ii)
`hybridising a primer to the template to form a
`template/primer complex,
`(iii)
`subjecting the primer to an extension reaction by
`the addition of compounds of Formula I with different
`nucleobases B corresponding the four bases A, C, T and G
`or analogues thereof,
`determining the type of the compound of Formula I
`(iv)
`added to the primer,
`(v)
`selectively hydrolysing the acetal protective
`group, and
`repeating steps (iii) to (v) sequentially and
`(vi)
`recording the order of incorporation of compounds of
`Formula I.
`The different compounds of Formula I in step (iii)
`may be added in sequence, in which case the four different
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`WO96/23807
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`PCT/SE96/00096
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`compounds I may carry the same label F. Alternatively, the
`different compounds I have different labels F and are
`added at the same time.
`In a preferred embodiment of the method of the
`invention, the template/primer complex is bound to a
`solid-phase support, such as a sequencing chip, for
`example. The template may be attached to the solid support
`via a binding linker, which, for instance, is ligated to
`the 5'-end of the template or incorporated in one of the
`ends of the template by polymerase chain reaction (PCR).
`The binding linker may then be attached to the solid
`support by use of a streptavidin coupling system.
`Alternatively, the primer may be attached to the solid
`support.
`The compounds of Formula I may, of course, also
`conveniently be used in so-called mini-sequencing (see
`e.g. Syvanen A-C et al., Genomics a:684-692 (1990).
`As is readily understood by a person skilled in the
`art, the compounds of Formula I may also be used in the
`synthesis of nucleotide chains, and another aspect of the
`invention relates to such use. For example,
`oligonucleotides and polynucleotides may be prepared by
`successively coupling compounds of Formula I to each other
`in any desired base order with intervening deblocking and
`using a non-template dependent polymerase, such as a
`terminal transferase. For such synthesis, the groups L2, Q
`and Fin Formula I may, of course, be omitted.
`The compounds of Formula I may be prepared by methods
`known per se. In a further aspect, however, the invention
`provides a particular method for the preparation of a
`subgroup of compounds of Formula I by direct 3'-0H
`protection, and more particularly compounds of Formula Ia:
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`Ia
`
`or a salt thereof, wherein B, X, Z, R1, R2, Q, F, m and n
`are as defined above, and p and q independently are
`integers from 1 to 10, preferably from 1 to 6, by reacting
`a compound of Formula II:
`
`X
`0
`0
`B
`II
`II
`II
`HO-P-O-P-O-P-0-00
`I
`OH OH OH
`
`I
`
`I
`
`II
`
`OH
`
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`or a salt thereof, wherein Band X are as defined above,
`25 with a compound of Formula III:
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`wherein R1, R2, Zand pare as defined above, and R3 is
`hydrocarbyl (for example, as defined for R1 and R2 above),
`to produce a compound of Formula IV:
`0
`0
`X
`B
`II
`II
`II
`HO-P-0-P-O-P-07Q
`I
`I
`I
`OH OH OH
`
`IV
`
`o,c_..zR1
`R2,..
`'\.
`(CH2)p
`'cooR3
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`WO96/23807
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`or a salt thereof, wherein B, X, Z, R1, R2, R3 and pare
`as defined above, optionally reacting the latter with a
`diamine H2N-.(CH2)q-NH2, wherein q is as defined above, to
`produce a compound of Formula V:
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`X
`0
`0
`8
`HO-P-O-P-O-P-0~
`I
`I
`I
`OH OH OH
`
`II
`
`II
`
`V
`
`o,c.,,ZR1
`R2,, '-
`(C~p
`,,.C ... N....-{CH2) q-NH2
`0
`H
`
`wherein B, X, Z, R1, R2, p and q are as defined above, and
`optionally coupling a dye label F to the terminal amino
`group.
`In the following, the invention will be illustrated
`by some non-limiting examples. Reference will be made to
`the accompanying drawings, wherein:
`Fig. 1 is a reaction scheme for the enolether
`synthesis described in Example 1 below. Intermediate and
`final products corresponding to various values of integer
`"n" in the respective structural formulae are identified
`by numbers indicated within brackets after then-values
`listed under the respective formulae.
`Fig. 2 is a reaction scheme for the preparation of
`3'-acetal-modified thyrnidine described in Example 2 below.
`Prepared final compounds corresponding to various values
`of integer "n" in the respective structural formula are
`identified by numbers indicated within brackets after the
`n-values listed under the formula.
`Fig. 3 is a graph showing thyrnidine 3'-acetal
`hydrolysis at pH 4 as log% of remaining acetal versus
`time in minutes.
`Fig. 4 is a reaction scheme for the one-pot synthesis
`of 3'-acetal protected deoxynucleotide triphosphates
`described in Example 4 below. Intermediate and final
`products corresponding to various values of integer "n" in
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`the respective structural formulae are identified by
`numbers indicated within brackets after then-values
`listed under the respective formulae.
`EXAMPLE 1
`Synthesis of enolethers for derivatization of nucleotide
`triphosphates (Fig. 1)
`step 1 Esterification of ketoacids
`The appropriate ketoacid (1 eq} was added at once to
`a large excess of dry methanol (20 eq}, previously treated
`10 with thionyl chloride (0.1 eq}. The homogenous mixture was
`refluxed overnight, evaporated under reduced pressure, and
`partitioned between saturated sodium hydrogen carbonate
`and dichloromethane. The combined organic extracts were
`dried with magnesium sulphate, evaporated and the residue
`15 was distilled under reduced pressure to give the
`appropriate methyl ester in a high yield (80% to 95%}.
`The following ketoacids were used:
`4-oxy pentanoic acid (levulinic acid} (Aldrich)
`1)
`5-oxy hexanoic acid has not been isolated. Instead a
`2)
`commercial ethyl ester-derivative of this acid (Merck) was
`transesterified in a reaction analogous to the above
`general procedure.
`3)
`6-oxy heptanoic acid was obtained from 2-methyl
`cyclohexanol (Merck), according to a published procedure
`(Org. Synth . .ll.: 3-5, (1951)).
`4)
`7-oxy octanoic acid was obtained from 2-acetyl
`cyclohexanone as described (J. Am. Chem. Soc. 1.Q.:4023-4026
`(1948)).
`step 2, synthesis of the dimethoxyacetal derivatives
`<compounds 1-4 in fig, ll
`Benzenesulfonic acid (0.01 eq) was added to each of
`above methyl esters (1 eq) dissolved in methanol (2 eq)
`and trimethylorthoforrnate (3 eq). The dark brown mixture
`was refluxed for 3 h, neutralized by addition of dry
`triethylamine (0.1 eq}, and evaporated. The residue was
`distilled under lowered pressure to yield a pure acetal.
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`Compound 1. The physical and NMR data for this acetal
`correspond well to the data reported previously.
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`Compound 2. Yield 85%, bp. 120° (15 mm Hg) ltt NMR (CDCl3):
`1.28 (s, 3 H), 1.60-1.69 (m, 4 H), 2.34 (t, 2 H), 3.17 (s,
`6 H), 3.67 (s, 3 H).
`
`Compound 3. Yield 92%, bp. 130-133° (15 mm Hg)
`ltt NMR
`(CDCl3): 1.25 (s, 3 H), 1.29-1.35 (m, 2H), 1.60-1.70 (m,
`4 H), 2.33 (t, 2 H), 3.17 (s, 6 H), 3.67 (s, 3 H).
`
`Compound 4. Yield 87%, bp. 147-149° (15 mm Hg)
`lH NMR
`( CDC 13 ) : 1. 2 5
`( s , 3 H ) , 1. 2 7 -1. 3 8
`( m, 4 H) , 1. 5 7 -1. 6 7
`4H), 2.31 (t, 2H), 3.16 (s, 6H), 3.66 (s, 3H).
`
`( m,
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`step 3, synthesis of enolethers {compounds 5-8 in Fig. ll.
`A mixture of the appropriate acetal (1 eq) and
`trimethylortoformate (0.5 eq) was placed in a distillation
`flask, equipped with a 20 cm Vigreux column.
`20 Benzenesulfonic acid (0.02 eq) was added and the mixture
`was refluxed. The heating was regulated so that the
`liberated methanol evaporated slowly. After 4 h the
`heating was increased and the dark mixture was
`fractionated under reduced pressure without previous
`neutralization of the acidic catalyst. The yields of
`isolated colorless enolethers were in all cases very high
`(85-95%) but GC and NMR analyses showed the presence of
`starting acetal in proportions from 10 to 30 %. Since
`these impurities were not expected to influence the
`derivatization of nucleotides no attempts were made to
`purify them further. The complete assignment of NMR
`signals was difficult because of the presence of starting
`material and the fact that these asymmetric enolethers
`exist as several isomeric forms. Nevertheless, in all
`spectra a vinylic signal of CH2 from one isomer at around
`4.4 to 4.5 ppm and a vinylic signal of CH from the other
`isomer at 3.85 ppm could be easily observed.
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`EXAMPLE 2
`Synthesis of 3' acetal-modified thymidine (Fig. 2)
`The 5'-protected thymidine, 5'-FMOC T (FMOC-fluorenyl
`methoxycarbonyl),
`(116 mg, 0.25 mmol) was dried by
`coevaporation with dry acetonitrile (10 ml) and dissolved
`in dry dioxane (5 ml). To this magnetically stirred
`solution an appropriate enolether (0.5 ml, = 10 eq)
`prepared in Example 1 was added, followed by
`trifluoroacetic acid (20 ml, 1 eq). After 45 min, the TLC
`analysis (Silicagel 60 F254, 10% methanol in chloroform)
`showed complete consumption of the starting material, and
`dry triethylamine (2 ml) was introduced in order to remove
`the base-labile FMOC-group. After this process was
`completed (60 min), the whole mixture was evaporated under
`reduced pressure. The nucleoside was separated from the
`excess of enolether by precipitation from petroleum ether,
`and the precipitate, dissolved in 5 ml of dry methanol,
`was treated with 1,3-diarninopropane (2 ml, 100 eq) at 60°
`for 180 min in order to effect the aminolysis of the ester
`function on the acetal moiety. Finally, the reaction
`mixture was evaporated under low pressure (oil pump) and
`the crude material was flash chromatographed on a silica
`gel column, equilibrated in ethanol and using a step
`gradient of cone. ammonium hydroxide (0-10%) in ethanol.
`The material, homogeneous by TLC (developed in 1-butanol -
`ammonium hydroxide 8:2), was combined, evaporated, and
`coevaporated with toluene, to give the NMR pure product
`(compounds 9-12 in Fig. 2) in high yield (72 to 85%). The
`pure material was stored as a stock solution in methanol
`after addition of three drops of ammonia.
`EXEMPLE 3
`Acidic hydrolysis (pH 4) of thymidine, substituted at the
`3 1 -position with different acetyl groups
`A reference acetate buffer pH 4.0, prepared by mixing
`of solutions of sodium acetate (0.20 M, 18.0 ml) with
`acetic acid (0.20 M, 82.0 ml) was used in all hydrolysis
`studies. To this buffer (10.0 ml) an ethanolic solution
`of the appropriate thymidine 3'-acetal derivative prepared
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`(100 ml) was added
`in Example 2 (compounds 9-12 in Fig. 2)
`with gentle stirring. At different time points a sample of
`0.5 ml was withdrawn and placed in a tube containing 15 ml
`of cone. ammonia to raise the pH to 9. Samples were
`analyzed by FPLC using an anion exchange column (Mono Q -
`Pharmacia Biotech AB) and a gradient system of
`tetraethylammonium bicarbonate pH 8.2 for elution (0.05 to
`0.75 M). The peak areas of starting material and its
`hydrolysis product thymidine were integrated, and plotted
`as shown in Fig. 3.
`
`to
`
`EXAMPLE 4
`One pot-synthesis of deoxynucleotide triphosphates,
`protected at their 3 1 -0H position by a functionalized
`acetal group (Fig. 4)
`A commercial deoxynucleotide triphosphate (pppdT,
`pppdC, pppdG, or pppdA) was chromatographed on a
`preparative Mono Q column using a gradient system of
`tetraethylammonium bicarbonate pH 8.2 (0.05 to 1.3 M)
`obtain the pure triphosphate in the form of a
`triethylammonium salt. This material was evaporated on a
`Rotavapor, coevaporated with dry acetonitrile (3x2 ml),
`and dissolved in molecular sieve-dried trimethylphosphate
`(0.5 ml). The appropriate enolether (compounds 5-8 in Fig.
`1) (0.2 ml) and trifluoroacetic acid (10 eq as a 10%
`solution in dry dioxane) were added. The homogeneous
`mixture was incubated at 20°C for 60 min, neutralized by
`the addition of triethylamine (100 ml), and precipitated
`from a 1:1 mixture of petroleum ether and diethyl ether.
`The oily precipitate was dissolved in methanol (2 ml) and
`1,3-diaminopropane (0.5 ml) or another diamine (1,4-
`diaminobutane, 1,6-diaminohexane) (0.5 ml) was added. The
`ester function was subjected to aminolysis overnight at
`60°c. The mixture was again precipitated from 1:1
`petroleum ether and diethyl ether, washed with diethyl
`ether, and dissolved in water. The water solution was
`analyzed and preparatively purified on the described anion
`exchange column. The well resolved products {compounds
`13-28 in Fig. 4) always appear prior to the original
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`deoxynucleotide triphosphate, having a retention time
`comparable to that of the appropriate deoxynucleotide
`diphosphate (as was found from separate coinjection
`experiments). An aliquot of the isolated 3'-acetal-
`5 modified derivative was evaporated, treated with 80%
`acetic acid for 2 min, and, after evaporation of acid,
`reinjected to the same chromatographic system. In all
`cases all starting material was reacted and a single
`product with higher retention time was formed that
`corresponded to the original deoxynucleotide triphosphate.
`Thymidine triphosphate reacts to form, besides the desired
`3'-modified derivative, also another product with even
`shorter retention time but that also hydrolyses to the
`starting triphosphate during acid treatment. It is assumed
`that this product is the bis-3'-0, 4-0-acetal-derivative
`of thymidine triphosphate. This type of side products was
`not present in reactions that used other nucleotide
`triphosphates. It should also be stressed that very little
`products of depurination were formed in reactions in which
`pppdG and pppdA were used. This can be explained by the
`mild acid applied as a catalyst, the large excess of
`enolether used in the reaction, and the fact that the
`bases existed in an unprotected (more acid resistant)
`fo