Field of the Invention

The present invention relates to methods for controlling the regio specificity and stereospecificity of enzyme catalysis with guide molecules. These methods may be used in biocatalysis for synthesizing both chiral and achiral chemicals. Moreover, guide molecules may be used in drug design for controlling metabolism of drugs and/or for deriving new prodrugs. The present invention also relates to novel methods for using either recombinant proteins or natural enzymes as crude, partially purified or in pure preparation to produce either precursors or final compounds with predetermined isomer structures.

Background of the Invention

Chirality plays an important role in the nature. Virtually all of the chemical compounds containing chiral centers have different functions in biological systems. This is due to the fact that enzymes and receptor proteins have different binding and/or substrate properties for separate isomers. By adjusting either chirality and/or structure of the drug it is possible to influence a biological system in a predefined way. This offers means to develop drugs to cure diseases or relieve the symptoms. In 1992, the Food and Drug Administration issued new guidelines strongly encouraging the development of single isomers as drugs. As a result, many new chiral drugs are being developed as single enantiomers. This has led to increasing need for chiral intermediates in large scale for the synthesis of pharmaceutical, industrial and agricultural chemicals.

The synthesis of optically pure chemicals is generally complicated. The resulting racemic mixtures of desired target compounds have to be further purified and isolated. In the field of synthetic chemistry guide molecules have been developed to change the equilibrium of the reaction to favor distinct isomer formation and 20% enrichment of a separate isomer is already a reportable result. Moreover, delicate crystallizing techniques may be used to isolate the desired enantiomer. Sometimes it is even possible to create such a synthesis route that provides only one single enantiomer, but generally

many techniques have to be combined to obtain satisfactory results. Enzymes are versatile natural catalysts having 1) chemoselectivity, 2) regioselectivity and 3) enantioselectivity as their natural properties. Thus, they may be used in conjunction with chemical synthesis, especially when chiral selection is required. Use of the enzymes as biocatalysts for preparing chiral chemicals is still novel and developing area of research.

Polyamine oxidase (PAO) from rat liver (EC 1.5.1.13) that preferably metabolizes N ¹ - acetylated polyamines (Fig. 1) was characterized by Hδltta in 1970's (Hδltta, 1977). He also showed that in the presence of certain aldehydes the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermidine or spermine as a substrate. This observation was later verified with PAO purified from porcine liver. Schiff base formation between primary amine and aldehyde, mimicking the structure and/or charge distribution of acetylated polyamine, has been suggested to explain the enhancement of reaction velocity of PAO. PAO has recently been cloned and further characterized. It thus offers a way to produce large quantities of recombinant proteins for several purposes.

The natural substrates shown in Figure 1 for PAO, namely N ¹ -acetyl- spermidine, N ¹ - acetylspermine and N ¹ , N ¹² -diacetylspermine, do not have any chiral centers. Therefore, PAO itself seems to lack stereospecificity with its natural substrates.

Spermine oxidase was initially characterized by Wang et al. (2001) and more adequately by Vujcic et al. (2003). It was shown to preferably metabolize spermine. Fmsl was characterized in 2003 by Landry and Sternglanz. PAO, SMO and Fmsl are all FAD-dependent oxidases having some common substrate properties but still clearly distinguishable from each other. Therefore, they are good candidates for studying the chemical regulation of enzyme catalysis.

Summary of the Invention

The present invention is directed to novel methods for controlling regiospecificity and stereospecificity of enzyme catalysis with guide molecules. The use of guide molecules in biocatalysis of different kind of chiral or achiral chemicals allows the production of chemicals with predetermined chiral/chemical structure. Guide molecules can be used to

destroy undesired isomers among the racemic precursor molecules (leaving the target isomer intact) or to produce selected precursor molecules by enzymatic cleavage of the guide molecule activated isomer in the racemic reaction mixture. Guide molecules may be used for controlling drug metabolism in humans/non-humans and also for preparing novel prodrugs.

Brief Description of Drawings

Figure 1. Guide molecule-controlled regio- and stereospecific enzyme catalysis by Fmsl, SMO or hPAO. Regiospecificity (El-3) and/or stereospecificity of enzyme catalysis could be regulated by using distinct guide molecules shown in the figure.

Figure 2. Natural substrates of polyamine oxidase (PAO). PAO catalyzes the degradation of spermidine and spermine derivatives, being acetylated by spermidine/spermine N^acetyltransferase, into 3-acetamidopropanal and putrescine and spermidine, respectively.

Figure 3. List of the abbreviations used for covalent guide molecule adducts.

Figure 4. Coding of the aldehydes used in the studies.

Detailed Description of the Invention

We have recently synthesized α-methylated polyamine derivates both in racemic and enantiomerically pure form, mimicking the functions of natural polyamines in cellular physiology and being metabolically stable. With these compounds we were able to show the dormant stereospecificity of PAO. Moreover, by using selected aldehydes we were able to regulate the stereospecificity. PAO with its complex features that allow the usage of aldehydes as guide molecules during enzymatic catalysis provides an excellent basis for developing novel biocatalysis methods for preparing chiral chemicals. Moreover, it may serve as a model to help us understand the chemical regulation of enzyme catalysis.

The present invention thus relates to a method for controlling the regiospecificity and/or stereospecificity of enzyme catalysis of FAD-dependent enzymes in preparing single isomer chemicals or achiral chemicals from compounds of formula

R ₂ R ₁ N- (CR ₃ R ₄ ) — N- (CR ₅ R ₆ ) _b — N- -(CR ₇ Rg)-N- -R ₉

R 10 R 11 R 12 n

wherein: each of a, b and c is an integer from 1 to 6; n is an integer 0 or 1 ; and each Of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R7, Rs, R9, Rio, Rn and R ₁₂ are, independently, hydrogen or alkyl of 1 to 6 carbon atoms; with the proviso that when n is 0, at least one of R ₃ , R ₄ , R ₅ and R ₆ is alkyl of 1 to 6 carbon atoms, and when n is 1, at least one of R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ is alkyl of 1 to 6 carbon atoms, and wherein the molecule has at least one chiral center.

The guide molecule has the formula

wherein:

Rl, R2, and R3 are, independently of each other, hydrogen, straight or branched, optionally substituted, optionally unsaturated C ₁ -C ₁₂ alkyl, an optionally substituted, optionally unsaturated C ₃ -C ₁₀ cycloalkyl, heterocyclyl, aryl or heteroaryl group, or Rl and R2 together with the adjacent carbon atom form a 3 to 10 membered saturated, partly saturated or aromatic ring, which may include one or two hetero atoms selected from N, O and S. The guide molecule is preferably an aldehyde. Preferable substituents Rl, R2 and R3 are those derived from the structures given in the table of Figure 4.

More particularly, the present invention relates as an example the usage of guide molecules to control regiospecificity and/or stereospecificity of enzyme catalysis of

human polyamine oxidase [EC 1.5.3.11] (PAO), spermine oxidase (SMO) or yeast Fmsl, not excluding the use of other similar enzymes and/or chemicals. A suitable enzyme for the purposes of the present invention is an enzyme which uses flavin adenine dinucleotide (FAD) as a cofactor and is susceptive to guide molecule control.

The present invention also relates to novel methods for preparing different kind of chiral or achiral chemicals with the aid of cell cultures, tissues, tissue extracts, bacteria and/or animals by using guide molecule-controlled enzymatic reaction.

The present invention also relates to novel methods for altering chiral/achiral drug metabolism in humans, non-humans and/or cell cultures originated from them with different kind of guide molecules to control the function of desired target enzymes and/or proteins.

The present invention also relates to novel methods to affect human or non-human physiology by the treatment with efficient dose of guide molecule or guide-molecule derived adducts to alter the function of target protein.

Explanatory Descriptions

A) ENZYME: Recombinant protein, protein source, target protein in human or non- human physiology, receptor protein (Fmsl), polyamine and spermine oxidases being given as examples, not excluding the use (or targeting) of other enzymes, structural proteins or receptors. B) PRECURSOR MOLECULE (PRODRUG; DRUG): (racemic, single isomer or mixture of both, and chemicals containing two or more chiral centers), normal cellular constituents. The precursor molecule may be a natural substrate of A or a compound that acts as a substrate after being attached to the guide molecule. Precursor molecule may act as an inhibitor and/or agonist/antagonist of A protein. C) GUIDE MOLECULE: Organic or inorganic compounds that affect the regiospecificity and/or stereospecificity of the target ENZYME; (aldehydes, not excluding the use of other chemicals that have ability to effect isomer/regio- or substrate specificity of A).

PAO = polyamine oxidase [EC 1.5.1.13]

SMO = spermine oxidase

Fmsl = Characterized as FAD-utilizing polyamine oxidase by Landry and Sternglanz (2003).

Materials and Methods For the used materials and methods the present inventors refer to the following scientific articles, which are incorporated herein by reference in their entirety: Jarvinen et al. (2006a); Jarvinen et al. (2006b); Grigorenko et al. (2005) and Jarvinen et al. (2005).

Examples

The invention is further demonstrated in the following examples. The examples presented herein are for the purposes of illustration only and are not intended to limit the scope of the present invention.

Example IA. Kinetic values of regiospecific cleavages of achiral spermidine by Fmsl with different aldehyde supplements [E ₁ (putrescine) and E ₂ (diaminopropane, DAP)]. The exact kinetic values have been determined with Lineweaver-Burk plotting using Graph Pad Prism 4.03 software with nonlinear fitting. K _cat values have been calculated assuming M _w of 58,833 for monomer and enzyme as a dimer consisting in one catalytically active center. Individual values and experimental conditions are shown in example IB.

Example IB. Regioselective cleavages of achiral spermidine by Fmsl.

The reactions were carried out in duplicate or triplicate at pH 9.0 in 100 mM Glycine- NaOH + 25°C. Recombinant Fmsl was added 1-2 μg/reaction mixture and the incubation time varied from 5 to 30 minutes. Linearity of reaction was monitored by

using Ti _/ 2 controls, i.e. samples that have been incubated for 2.5 to 15 min (half of the reaction time of an ordinary sample). Reaction mixtures without the enzyme supplement were used to control purity of the reagents and to exclude non-enzymatic degradation of the compounds. E ₁ cleavage was monitored by HPLC by measuring putrescine formation and E ₂ cleavage by determining DAP (diaminopropane) content as described in the publications listed in Materials and Methods.

Example 2. Regiospecific cleavages of chiral R- and S-MeSpd derivatives by Fmsl being supplemented with or without different aldehydes.

Increasing concentration of R- or S-MeSpd was used to monitor regiospecificity of Fmsl. Reference values for E ₁ and E ₂ cleavages of R- and S-MeSpd at 1 mM for below aldehyde supplements are marked bold. Fmsl 1 μg/reaction mixture, incubation time 15 min.

Kinetic values were determined from the above individual values with Lineweaver- Burk plotting using Graph Pad Prism 4.03 software with nonlinear fitting. K _cat values have been calculated assuming M _w of 58,833 and enzyme as dimer consisting in one active center.

The reactions were carried out in duplicate at pH 9.0 in 100 mM Glycine-NaOH +25°C. Kinetic values were determined with Lineweaver-Burk plotting using Graph Pad Prism

4.03 software with nonlinear fitting. K _cat values have been calculated assuming M _w of 58,833 and enzyme as dimer consisting in one active center. Fmsl was use at 1-2 μg/reaction mixture and incubation time varied from 10 to 30 min.

Example 3.

Regiospecific cleavages of covalently modified chiral α-MeSpd derivatives by Fmsl. Reaction conditions are explained in Example 4.

:iine

Ratio of k _{c a} , E ₁ divided by k _{c a} , E ₃ = 0.4

\^ _^ N - \^^^\/ ^/ '

Ratio of k _{c a t} E ₁ divided by k _{c a t} E ₃ = 15.80

Example 4.

The reactions were carried out in triplicate at pH 9.0 in 100 mM Glycine-NaOH +25°C. The amount of the enzyme protein, and incubation time are shown in the table. Kinetic values were determined with Lineweaver-Burk plotting using Graph Pad Prism 4.03 software with nonlinear fitting. K^ _t values have been calculated assuming M _w of 58,833 and enzyme as dimer consisting in one active center. Reaction velocities were about 80% in Glycine-NaOH compared to Tris-HCl at the same pH.

NA; (Bz-R-Me Spd) 4 μg/reaction 1 hour + 25 ° C, no reaction products detected.

N ^! Ac-Spd in triplicate 50, 100, 300, 600 and 1000 μM

Bn-R-MeSpd in triplicate 25, 50, 75, 100, 250,500 μM Fmsl 2μg/reaction mixture 15 min. Bn-S-MeSpd in triplicate 25, 50, 75, 100, 250,500 μM Fmsl lμg/reaction mixture 5 min.

Example 5. Degradation of covalent guide molecule adducts in rat liver homogenates.

100 μM drug equaled to 23000 pmoles of the analogue/mg of protein in the beginning of the reaction. A wistar rat was sacrificed and liver was removed and frozen with liquid nitrogen. Liver was homogenized at pH 7.4 in 25 mM Tris-HCl, ImM DTT and 0.1 mM EDTA buffer (1+3 w/v).Resulting homogenate was centrifuged at 13000 x g for 30 min at 4°C. Supernatant was directly used for biodegradation assay of covalently modified guide molecule adducts. A twenty μl aliquot of supernatant was added in 100 mM Glycine-NaOH pH 9.5, 5 mM DTT supplemented with or without 100 μM of studied drug in a total volume of 180 μl. After 10 or 30 min incubation at 37°C 20 μl of 50% SSA containing 100 μM DAH was added and assayed for products with HPLC as described in Materials and Methods. Data are average of two individual reaction mixtures. Protein content of liver supernatant was 39.2 μg/μl.

Example 6.

Degradation of racemic, (R)- and (S)-α-methylspermidine in rat liver extract in the presence of pyridoxal or benzaldehyde

Polyamine (pmoles/mg protein) putrescine spermidine spermine

2 h, 400 μM racemic MeSpd 301 ± 23 206 ± 27 212 ± 41 + 5 πoM benzaldehyde 2320 ± 70 166 ± 12 37 ± 3 + 5 mM pyridoxal 1170 ± 100 247 ± 14 63 ± 3 + 250 μM MDL72527 182 191 191

2 h, 200 μM (R)-MeSpd 261 ± 6 217 ± 6 169 ± 8 + 5 mM benzaldehyde 2550 ± 30 139 ± 24 21 ± 18 + 5 mM pyridoxal 350 ± 0 233 ± 12 55 ± 3 + 250 μM MDL72527 72 192 179

2 h, 200 μM (5)-MeSpd 221 ± 0 181 ± 35 172 ± 29 + 5 mM benzaldehyde 451 ± 69 158 ± 12 27 ± 6 + 5 mM pyridoxal 1450 ± 69 232 ± 23 49 ± 2 + 250 μM MDL72527 85 188 185

200 μM substrate equaled to 9060 pmoles of analogue/mg of protein in the beginning of the reaction. Reactions were carried out in triplicates (except MDL72527 reactions in duplicate) at +37°C for 2 h in 50 mM borate buffer at pH 9.3 containing 5 mM DTT, 100 μM pargyline and 1 mM semicarbazide. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. The duplicates differed less than 5 % from the mean. MDL72527, polyamine and spermine oxidase inhibitor; MeSpd, α-methylspermidine.

Example 7.

Kinetic characteristics of the degradation of spermidine, racemic and different isomers of α-methylspermidine by human polyamine oxidase in the presence of different aldehydes

K _m (μM) MO IC _e31 ZK _1n (S- ¹ M- ¹ )

AcSpd 14 8.5 610 x 10 ³

Spd NA NA NA

+ 5 mM benzaldehyde 9.4 0.85 79 x 10 ³

+ 5 mM P4CA 37 0.31 8.4 x 10 ³

+ 5 mM pyridoxal 25 0.49 20 x 10 ³

Racemic AcMeSpd 100 1.3 13 x 10 ³

Racemic MeSpd NA NA NA

+ 5 mM benzaldehyde 14 0.19 13 x 10 ³

+ 5 mM P4CA 55 0.11 2.0 x 10 ³

+ 5 mM pyridoxal 31 0.18 5.8 x 10 ³

(J?)-AcMeSpd 95 9.0 95 x 10 ³

(J?)-MeSpd NA NA NA

+ 5 mM benzaldehyde 20 0.68 34 x 10 ³

+ 5 mM P4CA 110 0.18 1.6 x 10 ³

+ 5 mM pyridoxal 5.9 0.01 1.7 x 10 ³

(S)-AcMeSpd 170 1.2 7.1 x 10 ³

(S)-MeSpd NA NA NA

+ 5 mM benzaldehyde 14 0.06 4.3 x 10 ³

+ 5 mM P4CA 44 0.09 2.0 x 10 ³

+ 5 mM pyridoxal 5.1 0.24 47 x 10 ³

The reactions were carried out at 3 to 6 different substrate concentrations ranging from 10 to 500 μM in duplicates at +37°C for 5 to 30 min in 100 mM glycine-NaOH at pH 9.5 containing 5 mM DTT and 0.05 to 2 μg of recombinant protein. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. NA, not applicable (30 min reaction with 2 μg of recombinant protein and 500 μM substrate resulted in less than 0.5 % degradation of the substrate); AcSpd, N ¹ - acetylspermidine; Spd, spermidine; AcMeSpd, acetylated α-methylspermidine; MeSpd, α-methylspermidine; P4CA, pyridine 4-carboxaldehyde.

Example 8.

Kinetic values of spermine and different diastereomers of bis-α,α'-methylspermine for human polyamine oxidase in the presence of different aldehydes

K _m (μM) kcat(S- ^J ') WK _1n (S- ¹ M ^"1 )

AcSpm 1.1 17 15 x 10 ⁶

DiAcNSpm 2.7 19 7.0 x 10 ⁶

Spm 47 0.4 8.5 x 10 ³

+ 5 mM benzaldehyde 4.5 12 2.6 x 10 ⁶

+ 5 mM P4CA 15 9.6 640 x 10 ³

+ 5 mM pyridoxal 4.1 2.9 700 x 10 ³

+ 1 mM pyridoxal 3.9 2.7 71O x 10 ³

Racemic Me ₂ SPm 47 1.0 21 x 10 ³

+ 5 mM benzaldehyde 28 5.8 210 x 10 ³

+ 5 mM P4CA 110 4.5 42 x 10 ³

+ 5 mM pyridoxal 23 2.0 87 x 10 ³

+ 1 mM pyridoxal 14 1.64 120 x 10 ³

(R^)-Me ₂ SOm 55 0.12 2.2 x 10 ³

+ 5 mM benzaldehyde 13 2.7 210 x Kr

+ 5 mM P4CA 170 0.53 3.0 x 10 ³

+ 5 mM pyridoxal 12 0.08 6.7 x 10 ³

+ 1 mM pyridoxal 21 0.13 6.2 x 10 ³

+ 5 mM benzaldehyde 21 4.5 210 x 10 ³

+ 5 mM P4CA 110 4.1 39 x 10 ³

+ 5 mM pyridoxal 6.6 1.2 180 x 10 ³

+ 1 mM pyridoxal 8.1 1.9 230 x 10 ³

+ 5 mM benzaldehyde 21 6.1 290 x 10 ³

+ 5 mM P4CA 330 7.8 24 x 10 ³

+ 5 mM pyridoxal 5.7 2.4 420 x 10 ³

+ 1 mM pyridoxal 27 6.0 220 x 10 ³

The reactions were carried out at 3 to 6 different (10 to 500 μM) substrate concentrations in duplicate at +37 °C for 5 to 30 min in 100 mM glycine-NaOH at pH 9.5 containing 5 mM DTT and 0.025 to 2 μg recombinant protein. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. AcSpm, JN^-acetylspermine; DiAcNSpm, N ¹ , N ¹ ^iacetylnorspermine; Spm, spermine; Me ₂ SPm, bis-α,α'-methylspermine; P4CA, pyridine 4-carboxaldehyde.

Example 9.

Putrescine production by human polyamine oxidase from racemic, (R)- and (S)-a- methylspermidine in the presence of pyridoxal, benzaldehyde or pyridine 4- carboxaldchydc racemic MeSpd (R)-MeSpd 36 (S)-MeSpd Ratio 72 nmoles nmoles 36 nmoles

Buffer system Supplemented with 5 mM benzaldehyde (R) I (S)

50 mM Na ₂ HPO ₄ pH 9.5 3.8 8.4 0.7 12

50 mM borate pH 9.3 6.7 8.5 1.1 8

100 mM glycine-NaOH pH 9.5 6.5 14.6 1.1 14

Buffer system Supplemented with 5 mM pyridoxal (S) I (R)

50 mM Na ₂ HPO ₄ pH 9.5 1.2 ND 2.3

50 mM borate pH 9.3 5.8 0.4 11.5 31

100 mM glycine-NaOH pH 9.5 3.5 0.2 9.0 50

Buffer system Supplemented with 5 mM pyridine 4- (R) I (S) carboxaldehyde

50 mM Na ₂ HPO ₄ pH 9.5 2.0 1.5 0.9 1.6

50 mM borate pH 9.3 0.4 0.3 0.1 1.9

100 mM glycine-NaOH pH 9.5 2.5 2.3 1.5 1.5

The reactions were carried out in duplicate at +37°C for 1 h in the indicated buffers containing 5 mM DTT and 1 μg of recombinant polyamine oxidase. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. No substrate degradation was detected in the absence of the enzyme. The duplicates differed less than 5% from average. MeSpd, α-methylspermidine; ND, not detected; Put, putrescine.

Example 10.

Effects of 17 aldehydes on the stcrcospccificity of human polyamine oxidase

Racemic MeSpd Λ-MeSpd S-MeSpd

Aldehyde % used

BA

^V^CHO 8.6 28.0 1.3

PL

CHO 11.6 14.6 8.2

P4CA

,CHO 6.3 20.0 1.1

MeO

5.8 25.4 0.7

/\^CHO 2.9 7.0 0.8

Of ,CHO 3.0 6.7 0.7

o 0.4 0.6 0.5

CHO

The percentages of the substrate used by hPAO in the reaction containing 200 μM racemic, (R)- or (5)-α-methylspermidine and different 5 mM aldehydes. Reactions were carried out in duplicate at +37°C for 1 h in 100 mM glycine-NaOH at pH 9.5 supplemented with 5 mM DTT, 5% EtOH and 1 μg of recombinant protein. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. No substrate degradation was found in the absence of the enzyme. The duplicates differed less than 5% from average. BA, benzaldehyde; PL, pyridoxal; P4CA, pyridine 4-carboxaldehyde; MeSpd, α-methylspermidine; ND, not detected.

Example 11.

Degradation of racemic, (R)- and (S)-α-methylspermine by human polyamine oxidase in response to different aldehydes

MeSpd Spd racemic MeSpm 1.2 1.1

+ 5 mM benzaldehyde 21.7 12.0

+ 5 mM P4CA 13.6 6.5

+ 5 mM pyridoxal 8.9 5.7

(J?)-MeSpm 0.8 1.9

+ 5 mM benzaldehyde ⁰ 13.6 22.3

+ 5 mM P4CA 3.2 9.6

+ 5 mM pyridoxal 3.1 2.0

(Sy-MeSpm 2.0 0.7

+ 5 mM benzaldehyde 26.9 5.7

+ 5 mM P4CA 21.5 5.5

+ 5 mM pyridoxal 13.5 8.7

500 μM substrate equaled to 90 nmoles in the beginning of the reaction. Reactions were carried out in duplicate at +37°C for 30 min in 100 mM glycine-NaOH at pH 9.5 containing 5 mM DTT and 0.2 μg of recombinant protein. Reactions were stopped and assayed for products with HPLC as described in Materials and Methods. No substrate degradation was found in the absence of the enzyme. The duplicates differed less than

5% from the average, a, 0.2 nmoles of putrescine was also detected; Spd, spermidine;

MeSpd, α-methylspermidine; MeSpm, α-methylspermine; P4CA, pyridine A- carboxaldehyde. It should be noted that MeSpm may be degraded from either end (Fig.

1).

Example 12. Covalcnt guide molecule adducts as substrates for hPAO

The reactions were carried out in duplicate in 100 mM glycine-NaOH at the pH 9.5 supplemented with 5 mM DDT. The amount of recombinant protein and incubation time are shown above. Kinetic values were determined with Lineweaver-Burk plotting using Graph Pad Prism 4.03 software with nonlinear fitting. K _cat values were determined using an M _w of 55,382 for recombinant human hPAO. The official name of the enzyme is PAOX polyamine oxidase (exo-N4-amino) M _w 55,382. Substrate range of 10, 25, 50, 75, 100, 200 μM with N ^! -AcSpd. Substrate range of 2.5, 5, 7.5, 10, 25 μM with Bz-R/S-MeSpd derivatives. Substrate range of 5.0, 7.5, 10, 25 μM with Bn-MeSpd derivatives.

EXAMPLE 13.

Degradation of α-mcthylatcd spermine analogs by polyamine and spermine oxidase with different aldehydes

PAO 0.2 μg SMO 0.3 μg

pmoles / 30 min in reaction mixture

MeSpd Spd MeSpd Spd

racemic MeSpm 1410 1410 9940 8920

+benzaldehyde 13000 7710 8970 10100

+pyridoxal 5160 3280 7150 7820

R-MeSpm 720 2170 880 1300

+benzaldehyde 11600 26400 1210 4110

+pyridoxal 2250 1460 790 1270

S-MeSpm 2240 890 23900 19800

+benzaldehyde 17540 3920 9690 8150

+pyridoxal 7730 4610 6980 7150

500 μM concentration of the substrate equals to 90'0OO pmoles in the beginning of the reaction. No spermine analog degradation in the reaction mixture was found in the absence of the enzyme. Reactions were carried out in duplicate in +37°C water bath for 30 min in 100 mM Glycine-NaOH pH 9.5 containing 5 mM DTT with or without pyridoxal or benzaldehyde. The duplicates differed less than 5% from average. Spd, spermidine; MeSpd, α-methylspermidine; MeSpm, α-methylspermine.

Example 14.

Degradation of bis-α-mcthylatcd spermine analogs by polyamine and spermine oxidase supplemented with different aldehydes

PAO 0.3 μg SMO 0.5 μg

pmoles / 30 min in reaction mixture

pmoles / 30 min MeSpd MeSpd

Racemic Me ₂ SPm 4630 11700

+ benzaldehyde 27100 10600

+ pyridoxal 9670 12000

RR-Me ₂ SPm 590 600

+ benzaldehyde 13300 2930

+ pyridoxal 520 1930

RS-Me ₂ SPm 6280 2300

+ benzaldehyde 31900 7490

+ pyridoxal 7290 3740

SS-Me ₂ SPm 4450 36100

+ benzaldehyde 34300 24300

+ pyridoxal 19100 9400

500 μM concentration of the substrate equals to 90'0OO pmoles in the beginning of the reaction. No spermine analog degradation in the reaction mixture was detected without enzyme. Reactions were carried out in duplicate in +37°C water bath for 30 min in 100 mM Glycine-NaOH pH 9.5 containing 5 mM DTT with or without pyridoxal or benzaldehyde. The duplicates differed less than 5 % from average. MeSpd, α- methylspermidine; Me ₂ SpIn, bis-α-methylspermine.

Example 15

Polyamine oxidasc-mcdiatcd stcrcospccific degradation of raeemic α- mcthylspcrmidinc in the presence of benzaldehyde or pyridoxal.

Reactions were carried out in duplicate at +37°C for up to 24 h in 100 mM glycine- NaOH at pH 9.5 containing 5 mM aldehyde, 5 mM DTT, 5 % EtOH and 1 μg of recombinant protein. Reactions were stopped and assayed with chiral-HPLC analysis as described in Materials and Methods. BA, benzaldehyde; D, 1,7-diaminoheptane; P, putrescine; PL, pyridoxal; MeSpd, α-methylspermidine; R; (7?)-α-methylspermidine; S, (5)-α-methylspermidine; Standard curve for raeemic MeSpd [(100, 200, 400 pmoles equaling to 50, 100 and 200 pmoles of single enantiomer; (7?)-MeSpd (r ² = 0.998; y= 526374x - 12048362); (5)-MeSpd (r ² = 0.999; y= 280813x - 6911740)] was used to determine (R/S)-enantiomer content in each sample. *, pmoles for (7?)-MeSpd calculated with pure (7?)-MeSpd enantiomer standard curve (r ² = 0.999; y= 336860x - 8595502).

References

Grigorenko, N.A., Vepsalainen, J., Jarvinen, A., Keinanen, T.A., Alhonen, L., Janne, J. and Khomutov, A.R. (2005). New syntheses of alpha-methyl- and alpha,alpha'- dimethylspermine. Bioorg Khim. Mar-Apr;31(2):200-5. Russian.

Hδltta E. (1977). Oxidation of spermidine and spermine in rat liver: purification and properties ofpolyamine oxidase. Biochemistry. Jan l l;16(l):91-100.

Jarvinen, A., Grigorenko, N., Khomutov, A.R., Hyvδnen, M. T., Uimari, A., Vepsalainen, J., Sinervirta, R., Keinanen, T.A., Vujcic, S., Alhonen, L., Porter, CW. and Janne J. (2005) Metabolic stability of alpha-methylated polyamine derivatives and their use as substitutes for the natural polyamines. J. Biol. Chem. Feb 25;280(8):6595- 601. Epub 2004 Dec 16.

Jarvinen, A.J., Cerrada-Gimenez, M., Grigorenko, N.A., Khomutov, A.R., Keinanen, T. A., Vepsalainen, J., Sinervirta, R.M., Alhonen, L. and Janne, J. (2006a) Alpha- methylpolyamines: Efficient Synthesis and Tolerance Studies in vivo and in vitro. The First Evidence for Dormant Stereospecificity of Polyamine Oxidase. J. Med. Chem., 49, 399-406.

Jarvinen, A., Keinanen, T. A., Grigorenko, N. A., Khomutov, A. R., Uimari, A., Vepsalainen, J., Narvanen, A., Alhonen, L. and Janne, J. (2006b). Guide molecule- driven stereospecific degradation of alpha-methylpolyamines by polyamine oxidase. J. Biol. Chem. 2006; 281(8): 4589-4595.

Landry, J. and Sternglanz, R. (2003) Yeast Fmsl is a FAD-utilizing polyamine oxidase. Biochem. Biophys. Res. Commun. Apr l l;303(3):771-6.

Vujcic, S., Liang, P., Diegelman, P., Kramer, D.L. and Porter, CW. (2003). Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion. Biochem J. Feb 15;370(Pt l):19-28. Wang, Y., Devereux, W., Woster, P.M., Stewart, T.M., Hacker, A. and Casero, R.A. Jr. (2001). Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure. Cancer Res. JuI 15;61(14):5370-3.