CA2382063A1 - Charge-modified nucleic acid terminators - Google Patents

Abstract

Charge-modified nucleic acid terminators comprising structure (I): Z-X-S-B-L wherein Z is mono-, di- or triphosphate or thiophosphate, or corresponding boranophosphate; X is O, CH2, S, or NH; S is a sugar or a sugar analogue; B is a naturally occuring or a synthetic base; L is alkyl, alkenyl, or alkynyl an d is optionally substituted with a reporter moiety; and L, B, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net positive charge to structure (I) at physiological or nucleic acid sequencing conditions. A method of sequencing nucleic acids using the above charge- modified terminators, as well as a method of inhibiting a virus which comprises contacting a cell infected with a virus with a virus-inhibiting amount of the above charge-modified terminator are also disclosed.

Description

CHARGE MODIFIED NUCLEIC ACID TERMINATORS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to United States Provisional Application Serial No.
60/154,739, filed on September 17, 1999, the entire disclosure of which is incorporated in its entirety herein.
BACKGROUND OF THE INVENTION
Field of the Invention The instant invention pertains to nucleic acid terminators which result in improved sequence data. The instant invention also pertains to charge modified nucleic acid terminators which allow for the direct loading of nucleic acid sequencing reactions onto separating media.
BACKGROUND OF THE INVENTION
The sequence of nucleotide bases in a DNA molecule can be determined in a variety of ways. The chain termination rriethod generally involves synthesizing DNA
complementary to the template strand to be sequenced by extending a primer able to hybridize to a portion of that tempiate strand with a DNA polymerase. During the synthesis reaction, deoxynucleoside triphosphates (dNTP's) are incorporated to form a DNA fragment until a chain terminating agent, for example, a dideoxynucleotide triphosphate (ddNTP) is incorporated. Incorporation ofa ddNTP prevents further DNA synthesis (a process called chain termination). The size of each DNA fragment synthesised in this procedure is then dete_rmir~d by gel el_ec_trophoresis and t_h_is informatio_n_ used to detere the seque_n_ce of nucleotides in the original template DNA. For example, Tabor and Richardson, U.S. Patent No. 4,99~,fi99, the entire disclosure c~f which is incc3rporated herein, describe a two step sequencing method in which an unlabeled primer is labeled in a labeling step, and then extended in the presence of excess dNTPs and a ddNTP in a chain termination step. In the labeling step, a 1Qw concentration of dNTPs is provided (one being labeled) to allow a small amount of primer extension.
In the dideoxy sequencing method, the primer may be labeled, for example with P3'', by a process using a polynucleotide leinase. Such labeling allows detection of extended primers a$er gel electrophoresis by autoradiography of the resulting gel.
Alternatively, a labeled d~lTP may be incorporated ~u~ng the process ~f I)NA synthesis, and the presence of such labeled dNTPs detected by autoradiography nr other means_ To this end, the dNTF may be labeled either radioacti~ly ~viti~ F32 ~r X35. In another Procedure,~the primer can be labeled with one or more fluuorescent xnuieties for dete~on by fluorescence.
In yet another procedure, the ddNTP may ba labeled, for example, with a fluorescent marker.
In a sequencing r~acti~, the te;~~s par~lly clece, most likely due to the thermocycling conditions, and generate labeled by-products which migrate in the separating media, thus ~t-erfermg with interpretation of the tie se~uene~g ~ragrneats.
For example, terminator d.ecampasitisn preductsand unreel tl;~minatars rnay appear an sequencing gels or electropherograms as peaks or blobs ~e.g., Figure 1, lanes 3 and 4 which show the blobs which result ~avhen sequencing reaction products contain~g ~onventl terminators are directly loaded onto an electrophoretic gel). At the prESent time, this problem is addressed by precipitation of the sequencing products using e.g., ethanol precipitation (e.g., Figure 1, lanes l and 2 which show gels which result ~rhen sequencing reactions containing conventional terminators are subjected to ethanol precipitation prior to being loaded onto an electrophor~tlc gel). ~Yh~e tlds races the ~~atic>n s~nev~hat, the procedure is tune consuming and creates a bottle-neck for any high through-put sequencing applications.
Thus, a process is weeded fur i~n~oving the clarity t~f seq. data. Such a process would not reqkaire additional sample prEparatiox~ steps. Id.eallyr such a process would reduce sample preparation time and result in improved seguencing through=put.
Moreover, such a method would also be economical to use. These and ot_h_er co_n_cerns are addressed in greater detail below.
~L~ O~' T~ II~~'fIL~hI
One aspect of the instant disclosure pertains to a charge-modified nucleic acid terminator comprising structure (I) ~X-S-~-L (I) wherein Z is mono-, di or triphosphate or thiophosphate, or corresponding boranophosphate X is O, CH2, S, or NH;
S is a sugar or a sugar analogue;
B is a naturally occurring or a synthetic base;
L is alkyl, alkenyl, or alkynyl and is optionally substituted with a reporter moiety; and L, $, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net posftive charge to structure (I) at physiological or nucleic acid sequencing conditions.
~n another aspect, the instant disclosure pertains tn methods of sequencing nucleic acids using the above charge-modified terminators. In yet another aspect, the instant disclosure pertains to methods of inhibiting a virus which comprises containing a cell infected with a virus with a virus-inhibiting amount of the shave charge-modified terminator.
I~ETA~i.ED I?~SGI~TION
The instant invention pertains to nucleic acid terminators, which along with the corresponding nucleic acid terminator decomposition products, migrate on separation media at di~ertnt rates than the sequencing reaction prsducts and which result in improved sequence data. Such nucleic acid terminators also allow for the direct loading of nucleic acid sequencing reactions QntQ separating rnedia. 'TQ achieve this goal, negatively Qr pQSitively charged moieties are attached to the terminator molecule. The unreacted or degraded terminators containing such charged moieties move faster (negatively charged) or in the reverse direction (positivEly charged) than the DNA sequencing products.
One embodiment of the charge-modified terminator according to the instant discl_osu_re is depicted in structure (I) Z-X-S-B--I, (I) wherein Z is mono-, di or triphosphate or thiophosphate, or corresponding boranophosphate X is t~, Cl-12, S, or NH;
S is a sugar or a sugar analogue;
B is a naturally occu_r_ring or a synthetic base;
L is alkyl, alk~nyl, or alkynyl and is optionally substituted with a reporter moiety; and L, B, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net positive charge to structure (~j at physiological or nucleic acid sequencing conditions.
The base may be any naturally occurring or synthetic base such as A, T, G, m' C or analogs thereof, such as 7-deazapurine, inosine, universal bases, etc.
Suitable base analogs include those disclosed in WO 99/Db422 and WO 97/28 ~ 77.
The sugar may be furanose, hexose, mono-di-triphosphates, morpholine, didehydro, didec3xyribose, deoxyribose, dioxalone, axathialane, and analogs thereof.
The linker may bE alkyl, alkenyl, or alkynyl and may contain 1 to about 100 atoms and may contains atoms such as C, H, N, O, S and halogen. In general, the linker contains about 2 to about 50 stems. Preferably, when the terminator molecule eantains a net positive charge, the linker contains about 2 to about 25 atoms, more preferably, the linker contains about 1 i to about 25 at<m3s. Preferably, when the term~ator ~lecule contains a net negative charge? the linker contains about 11 to 25 atoms, more preferably, the linker contains about 18 to about 2~ atoms.
The linker may optionally be substituted with a label, also referred to as a ''reporter or signal moiety.") The label may be a moiety such as a radioisotope, electrochemical tag, ~luarescea# tags, energy transfer {~T) labels, mass spectrometry tags, ltaman tags, haptea, chemilluminescent group, enzyme, chromophore, and two or more labels. ?h~
label may also be charged, e.g. ~y5.~, his-sulfonated carboxyfluorescein, or a dye attached to a charged moiety, e.g., carboxyfluareseein attached to cysteie acid or similar charged species. Methods for making these and other compounds are disclosed in U.S. Provisional Application No.
6~/El9$,4.b9 fled c~ august 31_, 199$, aid U,$, Appllcatio~ No, 90/01_ 8,69 _S
filed on Feb_rua_ry 4, 1998, and PCT/GB98/00978 filed on April 2, 1998 and published on October 8, 1998, the disclosures of each application incorporated in their entirety by reference herein.
The terminator molecule is substituted with a moiety which imparts a net negative charge or a net positive charge to structure (I) at physiological or nucleic acid sequencing conditiQ~s. The moiety _m__ay be a~~ charged species whic_h_ alters tl;e electrophoretlc mQbiiity of structure and degradation products, e.g., a-sulfo-j3-alanine, cysteie acid, sulfonic acids, carboxylates, phosphates, phosphodiesters, phosphonates, amines, quaternised amines, and p~spl~niu~ moieties. __A_s showy! i_n F-gore 2, ftte moiety (receded to as a "mobility modifier") may be attached to the terminator molecule bgtwg,~n the linker and label (Figure 2a), between the base and linker (Figure 2b), and may be attached only to the sugar (Figure 2g) or only to the linker (Figure 2 c). The terminator molecule may also contain multiple linkers and moieties which are alternatively spaced together {Figure 2d}.
Although the moiety may be attachBd only to the base, it is believed that the presence of a charged moiety at this position may affect adversely affect the reactivity of the terminator molecule. The moiety may also be made of a number of charged units covalently linked together.
The charge-modified nucleic acid terminators, which along with the corresponding nucleic acid terminator decomposition products, migrate on separation media at different rates than the sequencing reaction products and result in improved sequence data (i.e., no blobs which obscure true data) and permit direct loading of nucleic acid sequencing reactions onto separating media.
In particular, it has been found that the charge-modified nucleic acid terminators work especially well in sequenG~ng reacts which also co~ain golymerases such as ThermoSequenase, which is Taq A271/ F272M/F667Y. The full length enzyme was truncated to eliminate 5' to 3' exonuclease activity, and to provide a polypeptide more stable to proteolysis and heat treatment. Therefore position 1 (amino acid Met) in ThermoSequenase corresponds to position 272 in full length Taq polymerise.
Preferred polymerises include ThermoSequenase in which an amino acid substitution has been intradueed at E410 (the numbering is far ThermoSequenase, not far Taq palymerase). An especially preferred polymerise is ThermoSequenase containing an E410R, E410W, or E410M -s~bstit~tiQOS. Such polyme~ase~ are de~crzbed in PCTlUS00/221~0, the entire disclosure of which is incorporated herein by reference.
Additional preferred polymerises include a fuii length version of Taq polymerise with the following substitutions : D18A/E681R/F667Y. In this enzyme, the D18A
substitution removes the 5' to 3' exonuclease activity, rather than the deletion of amino acids as -i~ the The~moSesl~e~ase pc~lypeptide. The Efi$~lt. sub~titutiQ~n i~ the pQ~itiQn equivalent tQ
E410R in ThermoSequenase, and F667Y is the equivalent position to F396Y in ThermQSequenase. 'This enzyme ilso has properties desirable for sequencing with dye terminators. The amino acid sequence of Taq D18A/E681R/F667Y DNA polymerise.
Such polymerises are also described in PCT/CTS00/22150, the entire disclosure of which is i~co_rporated herein by reference.
The chard~-modified nucleic acid terminators may have other important applications.
They may also be useful in the therapeutic field as antiviral agents (anti-HIV
and anti-HBV

gtc~ (WO ~8149177~ and anticancer agents. Many nuclgosid.g and nucleotide analogues have been developed as antiviral agents. They o#len act by inhibition t~f 3?NA
polymerase and/or r~v~r~e transcriptase activity by a numbPa of means. A~ number of nucleoside analogues, such as ~T, ddC, ddl, D4'~, and 3?C are being used alone or in combination of other nucleoside or nc~ nucleo~de analogues as ~ti-~T ag~-s, The charge-rnodifred nucleic acid terminators ofthe present invention may also have antiviral activities alone or in combination with ether cc~c~mds, Sinoe cembinatie$ g therapy is being used more frequently to treat viral infections, having an increased number of compounds available by including ~fli~70~i3dS flf the present invention could enhance the possibility of successful treatments.
The ix~sta~ly ehazge_ifred terminators could be transp~ted into a cell in either the positively charged state, or if negatively charged, in a dephosphorylated state (which would then convert to a phas~c~rflat~d Mate ~ the ~ell~~r the ~laat~ groups could be masked to facilitate entry into a cell and the masking groups later removed. Oae embodiment of a charge-modified terminator according to the instant disclosure which may be used in antiviral applications is H.~-H H.N.H H.N.H
H H.N.H
1-i O J-~ O H O ~"~ O ~~ X) O
H'N'a-i .H?J'H ~-I'N.t-E - ~ O
H
The following examples are for illustration purposes only and should not be used in aay way to limit the appended el~ixr_~.

EXAMPLES
1. An example of charge modified reporters 1.1 Chemistry The following scheme was used to synthesize labeled ddNTPs with a charged reporter moiety. The linker was synthesized according to methods disclosed in U.S.
Provisional Application i~Io. 60/098,49 filed on August 31, 1998, the entire disclosure of which is hereby incorporated by referenee herein.
H H
H

N,H N~BSFAM N N N
I $~~ BSFAM BSFAM
I

O O O O

1~

QH eH OF,.I OH N~11-ddN~

H.N~O H.N~O +N~
H O~N,H O~N.H
H
H
HH

O O ' Rh d TFA Rhod o ~F~ _ Rhod = 5-8110, 5-ROX, 5-TAMRA, 5-REG

1.2 Discussion 4',3' Bis-sulfono-~-carboxyfluorescein was attached to 4-propargylamino-N-a-t-butoxycarbonylphgnylalaning by initial formation of the corresponding N-hydrt~xys~ccinhnide active ester using TSTU in DA~'iN,N-diisopropylethylamine.
Activation tines were typically 15 mines as observed by tlc before addition of the amino component. The product 1 was isolated by C18 Rf-HPLC then treated with neat tri#luoraacetic .acid to remove the carbamate moiety, with the product 2 isolated by Et24 precipitation. Attachment of the rhodamine dye was carried out using 5-rhodamine hydr~xysuccinhnde active esters in DAqSQfN,N-diisoprc~pylethylamine. All the double dye cassettes were purified by reverse phase HPLC prior to conjugation to alkylamino ddNTPs using published methods {and as disclosed in methods disclosed in U.S.
Provisional Application No. 60/098,469 filed on August 3 l, 199$, the entire disclosure of which is hereby incorporated by reference herein.). The labeled ddNTPs were purified by silica gel chromatography followed by ion exchange chromatography then reverse phase HPLC.
1.3 Experimental All chemicals were purchased from Sigma, Aldrich, Fluka or Fisher Scientific unless stated.
UV/visible spectra were rec.~rded ~n a Pe~rlEl~r_ L.ajmbda ~0 UV/vi~ihle spectrophotometer in conjunction with WinlabT~'~ software. Prep HPLC was car-vied out on a Waters LC 21100 or LC 4000 system on a C18 D~ltapak l5pm C18 100A SOx300mm column.
Ion exchange chromatography was carried out on a Waters LC 600 system.
~,propar~ylamido-4' S'-bissulfofluorescein)-N-a-t-butoxycarbon~ph~nylalaning (1) 4'-5'-bissulfo-S-carboxyfluorescein {IOOmg, 4.18mmo1) was dissolved in DMF
(4m1) then N,N-diisopropylethylamine (0.48m1, 15 eq.) and TSTU (65tng, l.2eq.) added. The reaction mixture was stirred at room temperature far 1h. then 4-propargylamino-N-oc-t-butoxycarbonylphenylalanine {b9mg, l.Oeq) added. Stirring was continued for 3h. then the reaction mixture evaporated to dryness in vacuo. The product was isolated by reverse phase HPLC {C18, DettaPak 15p., 100A, 50x3t10um) eluting with 0-100°~o eluant B over 60 min (A

= O.1M TEAB, B = 50°!° MgCNl0.IMTEAB vlv, 100m1/min.). The product (retention time 37 n3in.) was evaporated to dryness in vacuo then coevaporated with MeOH
(3x10m1) before lyophili2ation (100mg, 65°!°). UV/vis (1M triethylammonium bicarbonate pH 8.8) 495nm (24670), 465nm (shoulder, 9634), 312nm (6708).
~~roparg~lamido-4' S'-bissulfofluorescein~phenylalanine-a-ammonium trifluoroacetate 4-(propargylamido-4',5'-bissulfofluorsscein)-N-a-t-butoxycarbonylphenylalanine (100mg, 0.12mmo1) was treated with trifluoroacetic acid (lOml) for l5min. then evaporated to dryness in vncuo. The residue was coevaporated with toluene (3x10rnl) then the product precipitated by the addition of Et~O (50m1). The solid formed was collected by filtration, washed with cold Et2Q (3x50m1) then dried under high vacuum ( 100mg, 99°fo). Rf (tlc, iFrOH:NH40H:H~0 (6:3:1)=0.
General methodology for the attachment of rhodamine dyes to 2 (3) 4-(propargylamido-4',5'-hissulfofluorescein)-phenylalanine-o~-ammonium trifluoroacetate 2 {O.lmmol) was dissolved in DMSO (1m1) then N,N-diisopropylethylamine (0.26m1, 15 eq.) and rhodamine-NHS active ester ( 1.5 eq.) added. The reaction mixture was stirred at mom terx~eratuie for 16h, then evaporated to dryness in vacuo, The Rl 10, analog was treated with triethylammonium bicarbonate solution (0.1M, 50m1) for lbh to remove the trifluoroacetimido protecting groups. The products were purified by R)j-HPLf, using identical conditions to ~ unless stated. Retention times (BSFAM/R110 = 3lmin, BSFAM/R110 = 55min U-100°!° B over 90 min, 100 mllmin., BSF~.MIREG 54min 0-100°!°B
wer 9Q min., IQOmUmin, BSfAM/TAMR.A = 52min 0-1Q0°!° B aver 90 min). Au absorption spectra show the presence of both dyes.
General Methodology for Attaehment of 3 to alltynlamina-2' 3'-dideexYnueleotide triphosphates (4).

The double dye eassette(1D.0 p,mol) was dissolved inDMh {1m1) then disuccinimdyl carbonate {8 eq.) added as a solid at room temperature. The reaction was cooled to -60°C
then D{4 ~ in Due' {0.5~~ ceded. The r~ac~n mixture was warmed to -30°C
then a solution of aminoalkynl-ddNTl? {0.67gq., Na2C03/NaHCO~ pI~ 8.5) added. The reaction vas Stirred at room te~erature fcir 1h. then applied directly to a SiQ2 gei column. The product was Elrnted u~ i~'rOH:NHø01~:~20 (4:5:~ v:v:v) then evaporated to near dryness in uacuo before subsequent purification by ion exchange chromatography then C18 reverse phase HPI,C .as for 1. Absorption spectra of each compound showed the presence of both dyes.
1.4 Comparative E~eeh~ophero~r~ams figure ~ provides an example of the increase in migration rate relative to sequence products of unincorporated bis-sulfofluorescein energy transfer terminators (and thermal breakdown products thereof) ca~ared to the migratipn rate of the regular ET terminators.
2 An example of a ne a~; tivel~~ed linker arm 2.1 Background By incorporation of a number of charged amino acids into the linker arm it is possible to synthesise a labeled ddN'i'P containing extra negative charge that alters the mobility of the degradative by-products observed in a sequencing reaction.
2.2 Ch~mistr-y In order to determine the amount of negative charge required to remove colored by-products fron; the sequence ladder, ~luarescein vvas attached to oE-sulfo-~i-alanine to form 5.
Compound 5 was attached to 11-ddCTl? { 1 lumber of atoms in linker arm betw~n nucleotide and dye) to form 7. A portion of 5 was attached to a second a-sulfo-~-alanine lnQiety -to form 6 which was subsequently attached to 11-cldCTl' to form 8. A
control ddNTP

containing regular FAM attached to 11-ddCTP was also synthesized. The structures were run in a single color sequencing reaction to determine the effect of the charge on mobility.

H~N~Oti -~. fAI~I~N'~OH ~ FAiuI~N~N~Qti H S03--- H SO~- H SO~-!° 1' Q Q O O
Ns--11-ddGTP FAM'~N~N~N~11-ddGTP

As fluorescein carries a net 1- charge, compound 7 is considered as overall 2-linker arm, eampaund 8 has an overall 3- linker arm eharge.
2.3 Ex~~~r~mental N-5-carboxamidofluorescein-a-sulfo-~alanine f 5) a,-sulfo-~3-alanine (59mg, 0.35mmo1) was dissolved in DMF (2m1) then N,N-diisopropylethylamine (0.9mo1, l5eq) added followed by 5-FAM-NHS active ester (200mg, l.2eq.) The reaction mixture was stirred at room temperature for 3h. then evaporated to dryness in vacuo. The residue was coevaporated with MeOH ( l Oml) then the product isolated by C18 RP HPLC (A=0.1MTEAB, B=O.1MTEAB, 50°~oMeCN v/v) eluting with 0-100%B over 90 min at 100m1/xnin. 'H mnr (300MHz, CDsOD); 1.27(t, 24H, J=8.4Hz, NCHZCH,, _3.05(q, 16H, J=8.4Hz, NCH~CH3), 3.95-4.05(m, 3H, CH2+CHS03), 6.58(m, 3H, Ar-I~, 6.85(d, 2H, J=1 ~.OHz, Ar-I-~, 7.30(d, 2H, J=1 I.OH~, Ar-l~, $,02(x, 1H, J=7.6H~, ArH), 8.45(s,lH,Ar-H).
N-(N-5-carboxamidoffuorescein-a-sulfo-(3-alanine)amido-a-sulfo-~~(~alanine (6) N-S-carboxamidoffuorescein-a-sulfo-~3-alanine {5, 50mg, 0.095mmol) was dissolved in DMh (3m1) then N,N--diisopropylethylamine {0.25m1, l5eq.) and TST'~T (42mg, l.5eq.) added. ?he reaction mixture was stirred at room temperature for 1h, then a-sulfa-~-alanine (24mg, 1.Seq.) added. Stirring was continued for 3h. then the reaction evaporated to dryness in vacuo. The product was isolated by ion exchange chromatography (mono-Q column, A=0.1_M_ T-'CAB, 4Q°foI~eEN v/v, $=i.~A~'~'EAB, 4.tt1°feMeCN
v/v, 0-_50°foB over 22min_., 50-75%B from 22-SOmin. 75-100°!oB from 50-70 min., 4m1/min., retention time = 75-80min.) then CiB ~' Hl?LC {A=O.iM'1'EABB=-0.iM TEA$/MeCN 50°f° v/v, 0-100°f°B over 90 min., 100m1/min, retention time = 33min.). Rf (iFrOH6:ammonia3:waterlvlv/v) 0.34.
General Methodology for Attachment of modified dyes to alk~nlamino-2'.3'-didgoxynuclgosidg triphosphatgs (7,8).
The modified dye {lmmol) was dissolved in DMA' {5m1) then disuecinimdyl carbonate (4eq.) and DMAP (4eq.) were added at ~fl°C. The reaction mixture was stirred at -30°C for 15 min. then a solution of aminoalhyl-ddNTP {O.b7eq., NazCO~/NaHCO~ pH 8.5) added. The reaction was stirred at mom temperature for 1h. then applied directly to a Si02 gel column.
The product was eluted with ~rOH:NHaOH:H2Q (4:~: i v:v:v} then evaporated to dryness in vacuo before subsequent purification by ion exchange chromatography then C 18 reverse phase HPLC as for 1.
2.4 Results Each labeled ddNTF was used in a single color sequencing reaction using standard sequencing protocols to generate a sequence ladder. Interpretation of the electropherograms shown in Figure 4 provided the conclusion that an everall 3- charge i.e compound 8 removed the colored by-products from the electropherogram Figure 4 illustrates how the net negative charge of the clye labeled dideoxynuclec>tides e#fect~ their (and thermal breal~dQwn p~rQd~cts thereof] migration rate. As the n~t negative charg$ of th~ terminator incr~asgs, the migration rates of the various peaks seen (each of the peaks seen are either dye labeled dideoxynueleotides or thermal breal~down products thereof) increases (figure 4). At an overall 3- charge (2- from linker, 1-from fluorgscein) peaks are absent from the region of the electropherogram where sequence data would normally be obtained.
3 ~fie, ate-ively cl~.ed extended linker arms 3.1 Background In order to improve the effcieney of incorporation of the modified terminator, a labeled terminator with a 3- charge on the linker arm was synthesized, this time containing an extended linker arm of 18 and 2~ stems.
3.2 Ghemistrv p O O
FN~N~OH

O O O O
FAM N~N~1$~d~TP FAM~N~N~N~2~~~TP
H S03- H H SOs- H S03- H
g 10 3.3 E~rimeMtal Compound 6, was attached to 18-ddCTP and 25-ddCTP using the standard protocol for attachment of labels to ddN'fPs outlined in section 2.3. 'fhe method of purification was the same for 9 and 10.
Retention time of 9: Mono-Q ion exchange (47min) Retention time of 1D: Mono-Q ion exchange (42min) C 18 RP-HPLC ( 1 Smin) 3.4 Sequencing Results From the sequencing experiments it was clear that increasing the linker arm length improved incorporation of the terminator. This information, coupled to the presence of the 3- charge in the dye-linker structure led us to investigate rhodamine dyes with a 3-charged linker. This would permit four color sequencing. As shown in Figure 5, it is possible to directly load a sequencing reaction with no clean-up procedure. No peaks resulting from unineorparated dye-labeled terminator are observed in the sequence, demonstrating the utility of negatively charged with respect to direct load sequencing.
3 3 Rhodamine Labeled Terminators Containing a 3- Linker Arm The following chemistry was attempted to synthesize a four colored set of terminators H'N~OH --~Rhod~N~OH --~ ~~N~N~OH

x1,15, 1.g 12, 16, 20 O O O O O O O O
Rhod~N~N~N~OH ~ Rhod~N~N~N~N~X-ddNTP
ki S05- H S03-Ji S03- H S03- H S03 ki SO~- H
13, 17, 21 14, 18, 22, 23, 24, 25 TABLE I

Rhod ~ rhodamine label, ~ = length of linker arm, N~base 3.6 Ex,~erimental Compounds 11, 15, 19 were synthesized according to the method outlined for 5.
Compounds 12, 13,16,17,17,21 according to the method outlined for 6.
Compounds 14, 18,22-25 according to the general methodology for attachment of modified dyes to alkylamino- _ _2',3'-dideoxynucleotide triphosphates (7,$).
3.7 Results and Discussion The labeled triphosphates 14, 18, 22 were used in direct load sequencing experiment.
Compound 14 in a direct load experiment showed no breakdown products and with TSII and TaqERDAF'Y. Compounds 18 and 22 gave very dark sequencing bands and were observed to be forming an unexpected aggregate (as observed in the emission spectrum).
The compounds also produced large colored blobs on a sequencing gel which interfered with interpretation of the sequence.
In order to overcome the aggregation effect, structures 23-25 were synthesized to investigate the effect of a shorter linker arm. Compound 23 has been shown to yield a clean sequence.
4 Other examples of negativelX charged linker arms Other negatively charged linker arms have been synthesized and studied for example the phosphodiester structure shown below. The product was synthesized using phosphoramidite chemistry however it could also be synthesized via fl-phosphonates, phosphoroimidazolides, or phosphotriester chemistry.

FAM
o-Examples of energy transfer labeled ne ag'tivel~ged linker arm S.l Back~mound In order to increase signal intensity a range of negatively charged, energy transfer labeled nucleotides were synthesized.

5.2 ~'hemistru H
6iaRvFAM
H
O~N
IDI O'H
O
28 2~
CF,CC
~8 29 Rhod 3 SR110 30 Rhod = 5R8G
31 Rhod = 5TAMRA
32 Rhod = 5ROX
H
N.~FAM
H
H ~- H eo3 N
N~'H~'N~18-ddNTP
O_[O~~fIO
33 Rhod' SR11Q. N~
34 Rhod ~ 5R8G, N~U
35 Rhcd = STAMRA, N=A
39 Rhod ~ SRpX, N~
The sting material was synthesized according to the ET terminator patent and both e4 and ~
phenylalanine can be used in this ehemistry. The single dye amine aeid was reaeted with ec-sulfo-(3-alanine in DMp/DMSO to yield a compound of formal two minus charge.
The yield of compound 26 was improved by adding a,-sulfo-~i-alaning as a solution in DMSO.
Compound 26 was isolated by ion exchange chromatography and the previous reaction repeated to yield confound 27. The product was separated from starting material by ion exchange chromatography then the boc group removed by treatment with neat trifluoroacetic acid to yield compound 28. The rhodanune dye was introduced by reaction of the amine io aqueous buffer with a DMF solution of the rhodaming active ester. 'The products were isolated by ion exchange chromatography with any unreacted amine 2$ recycled from the ion exchange column. 'The charged energy transfer molecules were con3ugated to aminoalkynylnucleoside trzphosphates using the DSClDMAF method developed for energy transfer t~rn~ators and the products purified by silica gel chromatography, ion exchange separation then finally C 18 RP HPLC.
5.3 Experimental Boc-~nhenylalanine-4-(propar~ylamido-5-fluorescein)-a-sulfo-~3-alanine (26) Boc=~phenylalanine~4--(propargylamido(bis=pivaloyl)-5=fluorescein) (~OOmg, 0.~7mmol) was dissolved in DM~~' {4m1) then D1FBA ~~.25mi, 3eq.) added followed by Q-(N-succinimid3~l)-ICI,~I,N,~T'-tetrameth~leneuronium tetrafluoroborate ~ 1 S Smg, 1.1 eq.) in DMA' (1m1). The reaction was stirred at room temperature for Smin then a solution of a=sulfo=(3-ala~ne {95mg, i .2eq.) in DMSfl t3mi, dissolved by gentle heating) added at room temperature. The reaction was stirred at room temperature for lbh then evaporated to near dryness in vacuo. 'Phe residue was treated with N~,6H for 2h., the volume of the solution raced -in vuca~o, water (SDmI) added and the product p~rffied by ion exchange chromatography (Q sepharose) eluting with the gradient shown below. A = 0.1M
TEAB /
40% MeC~1 ~vwj, B = 1.0M TBAB ! 40°I~MeCAl {v!v) #lo~w = 6m~/min.
detect at 34Qnm,. (2.0 AUF'S). Product eluted in 50°!° buffer B.
?'ABLE 11 TimeJmin %B

2~ 10 4p 10 iiQ 4Q

TLC s;o2 Rf (iPrOH:NH4~H:H~O 6:3:1 v:v:v) = 0.45. 'H nmr (300 MHz, CD30I~);
1.39 (s, 9H, C(CH~) 2.27 {m, 2H, PhCH2), 2.80-2.85 (m, 3H, CHCH,, 3.45 (m, 1H, CHCHz), 3.75-3.85 (m, 3H, NHCH CHS03), 4.05 (m, 1H, CHCH2), 4.43 (s, 2H, propargyl CH,, 6.58 (s, 4H, FAM Hi', H1 ", H2', H2"), 7.0Q {ti, 2H, J=11.QH~, Ar-H), 7.2Q (d, 2H, J=11,OH~, phenylalanine Ar-H), 7.38 (d, 3FL, J=11.0>qz, 1 x FAM Ar-H,~ , 2 x phenylalanine Ar-H), 8.06 (d, 1H, J=7.6Hz, FAM H6), 8.45(s, 1H, FAM-H4).
N-( Boc-~phgnylalaning-4-(propar~~ylamido-5-fluorgscgin)-a-sulfo-~i-alaning)amido-a-sulfo=~3--alanine (271 Boc-~-phenylalanine-4-{propsrgylamido-5-fluorescein)-a-sulfo-~-alanine ( 100mg, 0.12 mmol) was dissolved in DMF (3m1) then DIPEA (O.lml, 3eq.) added followed by O-(N-succinimidyi)-N,N,N,N-tetramethyleneuranium tetrafluor~borate (50mg, 1. leq.) in DMF
{1m1). The reaction was stirred at room temperature for 5min then a solution of a-sulfo-(J-alanine {25mg, l.2gq.) in DMSO (1m1, dissolved by gentle heating) added at room temperature. The reaction was stirred at room temperature for 16h then evaporated to near dryness in vacuo. The residue was dissolved in water (50m1) and the product purified by ion exchange chromatography (Q sepharose) eluting with the gradient shown below. A
= O.1M
TEAB ! 40% MeCN {v/v), B = 1.0M TEAB / 40%MeCN (v/v) flow ~ 6ml/min. deteeted at 500nm,. (2.0 AUFS). Product eluted in 100% buffer B.
TAB1JE Hi _- Timelmin _ Q Q

75 ~0 12Q ~fl '~L~ s;~ Rf (iPrO~I:NH~,OH;H~O 6:3: i v:v:v) = 0,25. ~~I nn3r (30_ 0 MHz, ~CD30D); 1.39 (,~, 9H, C(CH~~ 2.27 (m, 2H, PhCH3), 2.80-2.85 (m, 3H, CHCH~), 3.45 (m, iH, CHCH~), 3.75-4.2D (m, fiH, 2xNHG1-~I C~iSQ3), 4.fl5 (m, iH, CHCHa), 4.43 (s, 2H, propargyl CH ), 6.58 (s, 4H, FA114 H1', Hl ", H2', H2"), 7.0D (d, 2H, J=i i.OH~, Ar-Hj, 7.22 {d, 2H, J=1 l.OHz, phenylalanine Ar-IT), 7.387 (d, 3H, J=1 l.OHz, 1 x FAM Ar--H,+ , 2 x phenylalanine Ar-H), 8.04 (d, 1H, J=7.6Hz, FAM H6), 8.47(s, 1H, FAM-H4).
'~i-t~henylalanine-4-(propargylamido-5-fluorescein)-a-sulfo-(3-alanine)amido-a-sulfo-f3-alanine (28) N-( Boc-~i-phenylalanine-4-(propargylamidQ-5-fluorescein)-a-sulfo-~3-alanine)amido-a-sulfo-~-alanine (iDQmg, D.i()mmol) was treated withtri#iuoroacetic acid (10m1) for 1h. then the reaction evaporated to dryness in va~u~. The residue was triturated with EtzO (30m1), the mother liquor was decanted from the yellow solid which was then dried under high vacuum.
General methodology for the attachment of rhodamine dues to 28 (29, 30, 31, 32) N-((~phenYlalanine-4-(propargylamido-5-fluorescein)-a-sulfo-(J-alanine)amido-a-sulfo- j3-alanine (20.Op.mol) was dissolved m NaHCO3/NazCO3 buffer (0.1M, pFI 8.5, 3m1) then the desired rhodamine-NHS active ester (1.5 eq.) in DMF (3m1) added. The reaction mixture was st3irred at rim terr~rat~~ fQr i Dh, then evapQrate~i 1Q diyne~~ in v~c~o.
T'lac X11 Q, analog was treated with triethylammonium bicarbonate solution (0.111, 50m1) for 16h to remove the tri#iuoroacetimido gratecting grflups then the product purified by ion exchange chron9atography. The other reaction m~~-tures were applied directly to a Q-sepharose ion exchange column and eluted with the following gradient. A = O.1M TEAB /
40°/s MgCN
{v/v), B = i.OM TEAS / 40°f°MeCN (v/v) flow = 6m11min. detected at 50Qnm, (2.0 AUFS).
Products eluted in 9D~° buffer $. Unreacted compound 28 eluted from the column in 100°~0 buffer ~ which could be recycled in later reactions.
TABLE IV
Time/min faB

so 90 Visible Absar~ptian Spectra data far ehar~ed ET cassettes 5lZi i0-(3F-SEAM-{as~3ala)2-OH 445nm (i.~2A), 5lQnm(0.72A) (TFA/H2O solvent) SRbG-(3F-_S~AM-(a,s~3ala)2-fl~-I 498nm (l.i4A), SZSnm (0.85A) STAM~A-~F'-SFAM-(cxsj3ala)2-OH 499nm (0.79A), 555nm (0.62A) SROX-(3>y-5p'AM-(as(3ala)z-OH 498nm(0.83A), 595(0.60A) General Methodology for Attachment of Modified Dyes to Alkynlamino-2'.3'-dideoxynucleoside Triphosphates (33, 34, 35, 36).
The charged ET cassette {16.D p,mol) was dissolved in DMF (1m1) then disueeinimdyl carbonate (8 ~q.) added as a solid at room temperatur~. The reaction was cooled to -60°C
then DMAP {4 eq) in DMF (0.5mi) added. The reaction mixture was warmed to -30°G then a solution of aminoallcynl-ddNTl' {0.67eq., NazC03/NaHCO~ pH 8.5) added. The reaction was stirred at room temperature for 1h. then applied directly to a SiQ2 gel column. The product was eluted with iPrOH:NH40H:H2fl (4:5: i v:v:v) then evaporated to dryness in vacuo bgfor~ subsequent purification by ion exchange chromatography then C 18 reverse phase HPLC as for compound 1.

5.4 Other due labeled. ne a~'vely charged nucleoside triphosphates Further negatival~ charged terminators have been synthesized using the chemistry outlined for single dye and energy transfer direct load terminators SRO ~F-SFAl~(as~iala)2-18-ddGTg (37) SFAM-(as~3ala)2-18-ddGTP (38) 5.5 U. Y. Visible Absorption Spectra of Compounds ~~-36 Ail samples were analyzed in TE buffer, pH 8.5. The absorption spectra of compounds 33-36 are found at Figures 6-~.
6. Examples of Terminators with Formal Fositively Charged Reporters 6.1 Eackground In order to study positively charged structures, the following labeled terminator was synthesized.
- H.N~N. 'v O
N. hl f N .r. NH2 O
Q ~ P,O
~P~O ø
a 6.2 Experimental Compound 39 (lOmg, 0.0134mmo1) was dissolved in DMF (1m1) then N,N-diisopropylethylamine (231, l0eq.) added followed by PyBOP (l4mg, 2.0eq.). The reaction mixture was stirred at room temperature for i5min. then a solution of 11-ddGTP
(0.0083mmo1, Na~CO~-NaHC03 pH 8.5) added in one portion. The reaction mixture was stirred at room temperature for 3h. then applied directly to a silica gel column. The product was ehrted with iPrDFI:N~OIi:H20 (b:3:1 v/v/v) then purified by ion exchange chromatography (as for 6) followed by C18 RP-HPLC (1.75~mo1 yield, 21%).
6.3 SeAUenein~ Results The electropherogram shown in Figure 10 was obtained when compound 40 was used in a sequencing reaction. The +2 charged terminator was used in a sequencing reaction and loaded directly on to a slab gel. The same experiment was repeated, however the reaction mixture was treated with phosphatase prior to loading on a gel to remove phosphates frflm the unincorporated dye-labeled dideoxynucleotides remaining in the reaction mixture. 'fhis would leave all terminator derived products with an overall positive charge causing them to migrate in the opposite direction to the sequence products during electrophoresis. It is clear from the electropherogram that the colored by-products are absent from the sequence when phosphatase is used to break down the terminator products.

7. Formal positive charged extended linker arms 7.1 Chemistry Another example of dyes attached to a positively charged linker arm is shown below;

~N~
~+, ~+, N~ Nw O H O
O ~ Rhod_.~.N N QI-I
H O
H? OH ~ ~~ OH
O N O
i In this example, the rhodamine dye R6G is attached to u-N,N,N-trimethyllysine which contains a formalized positive charge from the s quaternary amine. The product {41) can be further modified to yield a +2 linker arm {42) by reaction with a further molecule of the eharged amine aeid. Further reaetian(s) would generate the desired eharged strueture.
?.2 E,~ncrim~nta~
a-N~S~arboxamidorhodamine~Gy--s=N,N,N-_trimethyllysine 41~
~-N,N,N-~rimethyllysine {68mg, 30.Ommo1) was dissolved in L~MF {6m1) then N,N-diisopropylethylamine {O.SmI, l0eq.) added followed by R6G-NHS active ester (200mg, i.2eq.). The reaction r-nhctiue was stirred at room temperature for 16h: then evaporated to dryness in vacuo. The product was isolated by C18 RP l~llC {A=0_1M TAB, B=O.1MTEAB/50%MeGN, 0-100%B over 50 min., 100m1/min). Retention time = 44min.
a,-{a'-N-15-carboxamidorhodamine6G~-s'-N.N,N-trimgthyll~~ ~-N,N,N-trimethyll, s~ 42,~
a-N-(5-carboxamidorhodamine6G)-E-N,N,N-trimgthyllysine 28 (100mg, O.lSmmol) was dissolved in DMF (5m1) then N,N-diisopropylethylamine (0.3m1, l5eq.) and ~'S'~'U (f 7mg, l,Seqt) added. '~'he reactic_~r_i rnix_to_re was stirred at room terr~erature for 1h. t_h_en E-N,N,N-trimethyliysine ~SOmg, lSeq.) added. The solution was stirred for a further 3h. then the reaction mixture was evaporated to dryness in vacuo. The product was isolated by C18 RP

HI'LC (A=O.1M TEAB, B=O.1MTEAB/50%MgCN, 0-100°!oB ovgr 90 min., 100m1/min).
Retention time = 60min.
~ The Hse of Amino erroups in the Linker Arm as Carriers of Positive Char~ze 8.1 Background Compounds containing formal positive charge have numerous challenges associated with their chemical synthesis and purification. l~y using compounds containing amino functions, it may be possible to utilize profanation ef the amine moiety tc~ impart the desired charge.
The synthetic problems of formal positive charge may also be overcome by using a suitable lxotecting group to mask the amino funetion(s). The protecting group can be removed in a straightforward manner at the end of the chemical synthesis.
The initial molecule studied was lysine, which has a number of advantages.
2. The pRa ~f the s-NH2 function is approximately 10 hence the amino group will he charged at the pH ef a typical DNA sequencing separation 3. There are a number of protecting groups available for lysine which can be removed without degrading other moieties in the desired target mQleet~le 4. The number of lysines required can be readily modified using standard peptide synthesis methodologies (formal positively charged peptides containing s'-N,N,N-trimethyllysine are synthetically extremely demanding) A range of TAMRA labeled lysine oligomers were synthesized and conjugated to 11-ddATP
to determine the following information vVhether the amino group protonates under the conditions used in the separation step of dideoxy DNA sequencing?
How many lysine residues are required to prevent dye-labeled breakdown products from interfering with sequence information ?

8.2 Chemistry H=Lys,~OH ---~ H~Lys(TFA)},OOH -~ sTAMRA-lLys(TF~1}X=OH 6TAMR~-4--LysX-1l~idATP
43, jc=3 45, x=9 48, x=3 59, x=9 4-4, x=5 46, x=3 49, x=3 52, x=3 47, x--=5 50, x~5 53, x=5 Commercially available oli~olysines wer$ selectively protected at tl~ s-amino ~rou~ using the desired st~ichiometric amount of ethyl trifluoroacetate at 4°C.
g'he major product was isolated by C18 l~P-HP1JC and confirmed to be the desired product k~y~lectrospray mass spectrometry. The free amino groin was labeled using 6-T-NHS active ester then the ~r~d~t.attached tc~ 11 ~dATP g ~d ~an~u~at~on~he~stry. -C~rmpaunds 51, 52 and 53 were then used in a single color sequencing reaction and the products applied directly to a -sequencing gel {~i~ 37~j. A~ sk~c~wn -by the gel t~f Fire i i, increasing the number of line regi~dues remo~~es colored degradation byproducts fr$m the sequence.
Increasing the number of lysine residues is also believed to increase the reactivity of the substrate.
~'lrts ~ WI~Gh t~ ~~y~u~e ~9I;Cf~~'~ti~ Wa,,s l~pt G9n5tant aTld the ~n0ilnt of dideoxynucleotide concentration decreased showed that lower amounts of ddNTP
~;rm~ater~-continued tt~ eat longer sequencing ladders indicating that lysine modif ed ddNTPs are beer substrates for polymerase Than convEntional ddNTPs. Thus, it was concluded that the reactivity of the lysine ddNTPs increases in proportion the number of lysines present on the terminator molecule.
8.3 Experimental General method for introduction of trifluoracetamido protectin~,group to e--NHz of oligo---sine (46, 47) ~golysine tfl.l6mmol) was dissolved in MeQH (8m1) then triethylamine (1331, 6eq.) added ~d tie ~eactiort cc~aled to 4°C, ~1 trifluoroacetate f 3eq for 46, Seq for 47) was added and the reaction stirred at 4°C for 16h. The reaction was evaporated to dryness in a and the grc3duct purled by ~ 1 S ItPHPi.C using the gradient shown below.
The product containing fractions were evaporated to dryness in vaeuo and the product precipitated by the addition of Et~O (50m1). The mother liquor was decanted and the solid dried under high vacuum, Buffer A = Water/TFA (0.1% v/v),13 = Me(°:N/TF'A (0.1% v/v), 120 ml/min., 210nm detection.
TABLE V
T~~~ ~oB

General method for introduction of 6-TAMR.A to ~rotected~rsine xmers f48,49.501 Side chain protected lysine xmer (22.Opmo1) was dissolved in DMF {2 ml) then N,N---diisopropylethylamine (35,1, i0eq.) added followed by 6-TAMRA-NHS (lOmg, l.3eq.) in DMF {1m1). The reaction was stirred at room temperature for 16h. then the product purified by C18 RPHPLC using the gradient shown below. The desired product had the longest retention time on C i 8 HPLC in all cases. The product containing fractions were evaporated to dryness in vacuo and the product precipitated by the addition of EtzO
(SOmI). The mother liquor was decanted and the solid dried under high vacuum TABLE VI
Time/min %B

80 i00 Buffer A ~ WaterffFA (0.1% v/v), B ---- MeCNfTFA (0.1% v/v), 120 ml/min., 210nm detection.
General method for introduction of 6-~'AMRA to protected lysine xmers f51.52,53) TAMRA labeled protected lysines (48, 49, ~0, 3.9 ~mol) was dissolved in DMF
(3m1) then N,N-diisopropylethylamine {tl.3mi, 400ed.) d followed 1?y ~3-{N-succinimidyl)_ N,N,N',N'-tgtramgthylgnguronium tetraffuoroboratg (lOmg, lOgd.). Tl~ reaction was stirred at room te~erature for 5 min. then cooled to tl°~ before i 1-ddATP
(Smg} added in NaHCO3/NazC03 (0.1M, pH 8.5, 2m1}. The ice bath was removed immediately and the reaction stirred at room temperature for 30 min. I'he product was purified by silica gel chroaphy, eying with Me(~H to rive starting materials then 6:3:1 (~PrOH:
NH40H:H20 v:v:v) to elute the desired TFA protected compound. The product containing fractions were combined then evaporated tt~ year dryness i~ vacua and the product purified by mono-Q ion exchange chromatography eluting with the following gradient. A =
O.1M TBAB
/ 40°!° MeCN (v/v), B = 1.0M TBAB / 40°!°MeCN
(v/v) flow = 6m1/min. detected at 550nm, (2.0 ALTFS}.
TABLB ~I
Time/mtn fo$

90 i00 The product containing fractions were evaporated to dryness in vacuv then treated with NHaC?H ~50n~} for lbh. The reartic3n tta'e was evaporated to t~ryness then the product purified by C18 RgHfLC eluting with the following gradient. A = O.1M TEAB B =
lMeCN
flow = 2~m1/min. detected at ~~Onm, (1.0 AUFS).
TABLB VIII
Time%nin !oB -WO 01!19841 PCT/US00/25433 The product containing fractions were evaporated to dryness then the product dissolved in TE
buffer pH 8.5.
The methodology was repeated to synthesize the remaining three labeled nucleotides shown below SRiiQ-LysS-11-ddGTP (54) 6RbG-Lyss-11-ddLlTF (55) ~R~~-Lys3-11-dd~~'P (56).
A number of other dye-(lysine pentamer) nucleotides were synthesized to determine the effect of linker arm length and are shown below ~R~G-Lyss-18-ddUTP (5~) ~FAM-Ly~s6-11-ddGTP (~8) $ 4 FQUr c~loz~ ~e~ Pig u~~g tv~;ne labeled nucleotides In Figure 12., the sequence in lane 2 was generated using compounds 5~, 54, ~5, 56. As shown in Figure 12, unineorporated terminators eomigrate with sequencing data (lane 1), positively charged rhodamine terminators according to the instant disclosure migrate backwa_rd~ (lane _ - - - --- - - ---- - -- - - - -- - ---2), and negatively c_h_arged _rhoda_m__in_e terminators accor~iin~ to the instant disclosure migrate before sequencing data (lane 3).
8 5 Examples ofenergy transfer labeled polylvsine r~ueleotides The syntbesi~ of _h_exa~sin_e labeled nucelotide~ with an energy transfer label i~ ~hQwn below -~FAM

H~N O H~N O H-N O
O

~Qxf!1 IV N'~N N'_'~N N O
O O k1 H O ki O H O H
~OJIN OH
I ' Fi fll O N O N O
~F~-F H.F~F H F~F
F F
FAM M FAM
Q F F F ~ ~ F F F
F~F F~F F~F p~F p~F F~F
H.N O H.N p H'N.~O H.N O H.N O H'N O
O H O H O H O p p H O~ H O H O
~N~H H N~~ N~N N ~ ~~TAMRA N NY'N N~N N O
H H O ~H Q ~H Q H H H O H O ~ti O H
~,N O H.N O HN O ~ p .N O N O
'~ +f H - hf F~F F+F FF--~F F~F F~F F~F
F F

O
TAMI xN
~li O
w 62 O P O,, O O
~O
8. 6 Experimental a-t-butoxycarbonyl-(4-~rogar~ylamido-~-fluorescein~phenylalanine-hexa-(~-trifluoramido,~l rsine X59) a-t-butoxycarbonyl-(4-progargylamido-~-fluoresceinj-phenylalanine (~Omg, U.O~lmmol) was dissolved in DMF ~2~1) then N,N-dlisopr-opylethylamlne (90~i, iQ eq.) added followed by O-(N-succi~umidyl}-N,N,N',N'-tetramethyleneuronium tetraffuoraborate {23mg, 1.5 eq.} in DMF ~ 1 ml). The reaction was stirred at room temperature for 1 h. then further O-(N-s~ccit;dye)-N,N,~',~T'-tet;~~~a~i?tetro_roborate ($~g, 0,4eq.) added, The reaction was stirred at room temperature for a further 30min then the solution added to hexa-(a-trlfluoracetamidojiysine (77mg, l.Oeq.). The reaction was stirred at room temperature for ~f~, then t_h_e ~'od~cl i~lated by C1$ ~~~ eating with the gradient as ~_h_Qwn ~elQw. The product containing fractions were evaporated to near dryness in uacuo, then the suspension frozen and lyophilized (3img, 30°f°).
Buffer A = waterITFA (0.1°!o v/v}, B = MeCN/TFA (0.1°!o v/v), 120 ml/min., 210nm and 445nnideteetiog. - -Product eluted at t=50 min. TOF MS ES+ found 2023.0 (M+Na+), theoretical C86HioiFisNi44zz 2023.7.

Time/min !o$

gp 100 ~4-t3ro~argyylamido ~ fluorescein~phenylalanine-hexa-(s-trifluoramidollysine (60) ec-t-~t~xycarbonyi-(4-prQgargylarnidQ-5-~resee~)-nylalaniae-hexa-(~_ trifluaramida)lysine X59, 30mg, 14.41uno1} was treated with trilluaroaeetie aeid ( l Oml) far 30 min. then evaporated to dryness in vacuo. The residue was treated with EtzO
(SOmI) to afford a yellow solid. The Supernatant liquor was decanted, the residue triturated with further portions of Et2D (2 x l Oml} then. dried under high vacuum. TDF MS ES+ found 1944.7 (M+Na*), theoretical CalH9iF1aN1a8zoNa 1944.6.

a N STAMRA-4-Qro~argylamido-5-ffuorescein)-phenylalanine-hexa-(e-trifluoramido)lvsine (4-progargylamido-5-ffuarescein)-phenylalanine-hexa-(~-trifluoramido)lysine (60, 100mg, 49.Op.mo1) was dissolved in I~Mh' (1m1) and N,N-diisopropylethylamine (851, l0eq.) added.
The solution was added to a solution of 5-TAMRA-NHS (SOmg, 1.9eq.) and the reaction ~nixtu~~ ~t~~d al _roQ_m__tem~e_r~ture for 1_6h. T_h_e product was purified by C18 RP _H_PL~, by elution with thg following gradient;
Buffer A = water/TFA {0. i °fo v/v), B = MeCN/'TFA (0.1 °!o v/v), 120 m1/min., ~~Onm and 445nm detection. Product eluted at t=50 min.
TABLE X
Time/min /'nB

_ The product eluted at 7~ min and was observed to have absorptions at 44~nm (lactone form of ffuoreseein) and ~30nm(TAMRA absorption). The product containing fractions were combined and evaporated to dryness in uacuo and the residue triturated with Et20 (100m1) before drying under high vacuum. TQF MS ES- found 238.4 (M-~, theoretical Clo~HmF'igN1642a 2328.7.
TANiRA-(4-progargylamido-5-ffuorescein~»hen~alanine-hexalysine-11-ddATP
coniu~ate a-N-STAMRA-4-progargylamido-S-fluorescein)-phenylalanine-hexa-(s-trifluoramido)lysine (61, lOmg, 4.3~mol) was dissolved in I~MF ( 1m1) then N,N-diisopropylethylamine (7p1, l0eq.) added followed by O-(N-succinimidyl)-N,N,N,N'-tetramethyleneuronium tetraffuoroborate (6mg, Seq.). The reaction mixture was stirred at room temperature for ~

min. then cooled to 0° C before addition of 11-ddATP (0.3m1, 4mg, 1~ in NaHCO~/Na3C03 {phi 8.5) buffer. The reaction was raised to room temperature and stirred for a further 1h.
The product was purified by Q sepharose ion exchange chromatography using the gradient shown below;
Buffer A ~ O.1M TEAB/MeCN (40% v/v), B ~ 1.0M TEAB/MeCN (40% v/v), 120 ml/min., SOOnm and 550nm detection. Product ehrted at t=38 min.
TABLE ~
T~~ foB

The product containing fractions were evaporated to dryness in uacuo and the residure treated with N~DH {e. l Oml) far 16h. The reaction mixture was evaporated to near dryness in vacuo then the product repurified by Q sepharose ion exchange chromatography using the same gradient. The product containing fractions were evaporated to dryness in vacuo then dissolved in TE buffer prior to use in a sequencing reaction.
8.7 FrllL~l~O~ ~y~6-~~-ddCT~'synthesis FAMROX-Lys6-11-ddCTP was synthesised using the following pathway:

alpha andag cf 59 ----~ ---~ " _~ f ~ i ~ y i ~ ~l .»~.N.. "...~"
Compound 59 was attached to 11-ddCTP using O-(N-succinimidyl?-N,N,N',N'-tetramethyleneuronium tetrafluoroborate as the activating reagent. The product was deprotected as a crude mixture using neat trifluoroacetic acid for 20 min.
then any unreacted 11-ddCTP removed from the reaction by Cl$ I~PLC. The modified nucleotide was then dissolved in DMS~ and reacted with a DMS~ solution of 5-R4X-NHS active ester.
8.9 Experimental (4-pro~gylamido-S-fluoresceinj-phenyialanine-hexa-(s-trifluoramido)lysine-1 i-ddGTP
canju~ate (63) oc-t-butoxycarbonyl--(4--progargylamido--5-fluorescein)-phenylalanine-hexa--(s--trifluoramido)lysine (~9, 30mg, i~Opmol) was dissolved in DMF (1m1) then N,N-diisopropylethylamine (251, k(?eq.) added followed by C?-(N-succinimidyl)-N,N,N',N'-tetramethyleneuronium tetrafluoroborate ( l Omg, 2eq.) in DMF (O.~rnl). The reaction was stirred at room temperature for 10 min., cooled to 0°C then 11-ddCTP
(DMF solution, 5.$mM, 3m_1, 1_.2eq.}added. The solut~y. was _raised to room temperature and stirring °' U o continued for 3h. The reaction was evaporated to dryness in vacuo and the residue trituratgd with EtzO (Stlml). The supernatant liquid was decanted and the solid dried under high vacuum. The residue was treated with TF'A (15m1) far 20 min then evaporated to dryness ~~
vacuo. The residue was dissolved in water (100m1) and remaining unreacted 11-dCTP
removed by C18 HPLC. The product containing fractions were combined and evaporated to dryness in vacuo.
Buffer A = water/TF'A {0.1 °~o v/v), B = MeCN/TF'A (0.1 °!o v/v), 120 ml/min., ~ OOnm and 445nm detection. Product eluted at t = 50 min.
TABLE ~I
Time%in foB

5-ROX-(4-progargylamido-5-fluorescein~nhenylalanine-hexalysine-11-ddCTP
conjugate (4-$rogargylamido-5-fluorescein)-phenylalanine-hexa-(E-trifluoramido)Iysine-11-ddCTF
conjugate (63, approx. l5~mol) was dissolved in DMSO (3m1) and N,N-diisopropylethylamine (52p1, l0eq.) added followed by 5--RO~-NHS (47mg, 75~mo1) in DMSQ {5n~). 'tee reacxien was stirred at-room temperature for i~h. then the protected protected nucleotide purified by C~-sepharflse ion exchange chrflmatography eluting with gradle~rt A shown below followed by ~ l 8 RP-1-PLC (gradient B). The desired fractions wrxe evaporated to dryness in vacm then the residue trued with NHdOH (e., 200m1) far 16h. The reaction was concentrated to near dryness in vacuo then the product purified by Q
sepharose ion exchange chromatography eluting with gradient A.
gradient A, Bu#fer A = fl. l M TEAB/MeCN (40°~o v/v), B = 1.0M
TEAB/MeCN (40°fo v/v), 120 ml/min., 500nm and 575nm detection. Product eluted at t=50 min.
TABLE XIII

T~~~ %B

Gradiern B, Buffer A = O.1M TEAB, B = MeCN, 120 ml/min., SOOnm and 575nm detection.
Product glutgd at t = 38 min.
TABLE XIV
%B

~.1 D Ll. Y. Visible Spectra of Compounds 62 acrd 64 Samples were analyzed in TE buffer, pH 8.5. LTV/visible spectra for compounds ~2 and 64 are shown in Figures 13 and 14.
9 Example of reporterless dideoxynucleotide d~hos~hate bearing_twsitivel~ eg-dd groups Synthesis of H-Lys6-11-dcICTP f651 H Lysb-11-ddCTP was synthgsi~d as follows:
W
""~,P,~.~o~m_.
h Q11F
N
.e, o ..P~O O_ 4 ~P.
O
'fo a clean, dry vessel was added $oc-(Lys(TFA))6-Ql-I (33mg, 22.SS~moles) and this was dissolved ~ ~MF (2m~j. To this was added ~'S~'U as a Dl_1~' solution (13.6m~, 45.OS~moles in 1m1) and D~PEA (0.226mmoles, 39,1). 'The vessel was swirled several times to ensure a colorless solution. After 1 hour, 11-ddCTP, as the trigthylammonium salt, was added as a DMF solution (22.SS~moles, 3.9m1) and the solution swirled several more times.

3 hours after addition of ddNTP the reaction was reduced to a clear gum in vacuo. Addition of diethyl ether (20m1) and vigorous swirling caused the gum to form into flakes of white solid. T'he ether was then removed via pippette and the white flakes dried in vacuo. The flakes were then dissolved in neat trifluoroacetic acid { lOml), the solution swirled and left for 20 minutes. The acid was removed in vacuo to leave a clear oil, and more ether was added (20m1) to farm white flakes of solid. This solid was dissolved in I?MF (lOml) and loaded onto a preparatory Clg I4FLC system with monitoring at 300nm and a flow rate of 100m1/miw TABLE XV
Time (rains)%A (Water 0.1% v/v TFA) %B (Acgtonitrile 0.1%
TFA) o loo 0 Peak eluting between 47 and 50 minutes was collected and reduced to dryness in vacuo.
Acetonitrile (SOmI) was added and the mixture reduced to dryness again. This was repeated 2 more times until a white solid remained. Trituration was effected with ether and then the liquid removed via pippette. The solid was dried in vacuo before ammonium hydroxide was added (100m1). The solid did not immediately dissolve and so the mixture was stirred vigorously overnight. The next day, a clear solution remained which was then reduced to approximately one third volume in vacuo. Normal phase tlc of the remaining solution exhibited a 254nm spot, ~f= 0, with a mobile phase of 6:3:1 iP~rQ~I : N~4Q~ ;
~IzQ, Solution loaded onto APBiotech MonoS ration exchange column X10 with deionised water. Monitoring at 300nm and flow of ~mlmin-'.
Time {rains)%A (0.1M TEAK 4t1! fB {iM TEAB 40! v/v Acetonitrile pfl 7-8) Acetonitrile pfI 7-8) i0Q 0 Peak at 47-51 minutes collected and reduced to a clear gum in vacuQ. Methanol (25m1) was added and the mixture reduced again. 'Phis was repeated twice further until a white solid remained. The white solid was dissoved in p~ ~.S Tris-E~'~'A buffer (2m1).
Itield ~.$4~~nQles ($.2°fQ).
~lectrospray mass spectrometry, positive ion mode: CS~H~p3~T17~19P3 ~Z where ~2, 693.1, monosodium salt CsøPiiQ3NmNa0isP3 m/z where ~2, 704.6, trisodium salt C~4HiaNmNa3419P3 m!2 where ~2, 726.1.
Abbreviations Abbreviation Definition ddNT P 2'-3'-dideoxynucleoside triphosphate ET Energy Transfer TSTU 2-Succinimido-l,1,~,3-tetramethyluronium tetrafluoroborate PyB4P Benzotrialzol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate DMF N,N-dimethylformamide RP HPLC Reverse Phase High Performance Liquid Chromatography Et2Q Diethyl her DMSO Dimethyl sulfoxide 'rEAB Triethylammonium bicarbonate M~GN AcctQn~rile iPrOH lsopropanol NH40H Ammonium Hydroxide BSFAM 4',S' Bis-sulfono-S-carboxyfluorescein 8110 Rhodamine 110 REG or R6G Carboxyrhodamine6G

TAMRA Tertamethylrhodamine ROX Carboxy-X-rhodamine DMAP 4-dimethylaminopyridine i i-ddGTP 2',2'-dideoxyguanosine triphosphate with an 11 atom linker arm NHS N-hydroxysuceinixnide Although various embodiments of the instant invention are described in detail above, the instant invention is not limited to such specific examples. Various modifications will be readily apparent to one of ordinary skill in the art and fall within the spirit and scope of the following appended claims.

Claims

What is claimed is:

1. A compound comprising structure (I) Z-X-S-B-L (I) wherein Z is mono-, di or triphosphate or thiophosphate, or corresponding boranophosphate X is Q, CH2, S, or NH;
S is a sugar or a sugar analogue;
B is a naturally occurring or a synthetic base;
L is alkyl, alkenyl, or alkynyl and is optionally substituted with a reporter moiety; and L, B, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net positive charge to structure (1) at physiological or nucleic acid sequencing conditions.

4. The compound according to Claim 1, wherein the moiety which imparts a net negative charge or a net positive charge to structure (I) is selected from the group consisting of .alpha.-sulfo-.beta.-alanine, cysteic acid, phosphate, sulfate, sulfonate, carboxylate, phosphodiester, phosphonate, phosphonium, amine, and higher alkyl or aryl amines.

5. The compound according to Claim 3, wherein the moiety is a primary, secondary, tertiary, or quarternary amine.

6. The compound according to Claim 3, wherein the moiety is lysine.

7. The compound of Claim 1 wherein L is substituted with a reporter moiety.

8. The compound of Claim 1 wherein the reporter moiety is an energy transfer label.

9. The compound of Claim 1 wherein the linker contains up to abut 100 atoms.

10. The compound of Claim 1 wherein the linker contains about 2 to about 50 atoms when structure (I) contains a net positive charge.

11. The compound of Claim 1 wherein the linker contains about 11 to about 25 atoms when structure (I) contains a net positive charge.

12. The compound of Claim 1 wherein the linker contains about 11 to about 25 atoms when structure (I) contains a net negative charge.

13. The compound of Claim 1 wherein the linker contains about 18 to about 25 atoms when structure (I) contains a net positive charge.

14. A compound selected from the group consisting of wherein L is an alkyl, alkenyl, alkynyl containing mare than ten atoms when N
is a purine base or analog thereof and L is an alkyl, alkenyl, alkynyl containing more than twenty atoms when N is a pyrimidine base or analog thereof;
and isomers thereof.

15. A compound comprising structure (VI) wherein Rhod is selected from the group consisting of 5R110, 5R6G, 5TAMRA, 5ROX and isomers thereof;
L is an alkyl, alkenyl, alkynyl containing more than four atoms, and isomers thereof.

16. A compound selected from the group consisting of and isomers thereof.

17. A method for sequencing a nucleic acid that comprises:
A. reacting a nucleic acid template, nucleoside triphosphates or analogs thereof, and compound (I) according to Claim 1 with a polymerase to generate fragments, B. electrophoretically separating said fragments, and C. determining the sequence of the nucleic acid template.

18. A kit for sequencing nucleic acids comprising compound (I) of Claim 1.

19. A composition comprising compound (I) of Claim 1.

20. A method for inhibiting a virus that comprises contacting a cell infected with a virus with a virus-inhibiting effective amount of the compound of Claim 1.

21. A compound of formula XI
and isomers thereof.

22. A method for inhibiting a virus that comprises contacting a cell infected with a virus with a virus-inhibiting effective amount of the compound of Claim 21.

23. The method according to Claim 20 wherein the compound of Claim 1 is administered orally, bucally, topically, intravenously, parentally, or rectally.