ISSN 0012-5008, Doklady Chemistry, 2006, Vol. 410, Part 2, pp. 170–173. © Pleiades Publishing, Inc., 2006.Original Russian Text © V.I. Lozinskii, I.A. Simenel, A.R. Khokhlov, 2006, published in Doklady Akademii Nauk, 2006, Vol. 410, No. 4, pp. 487–490.

170

Computer design of so-called protein-like copoly-mers whose macromolecules are constructed from twotypes of monomer units with different hydrophilic-ity/hydrophobicity and whose heteroblock chains havea conformation similar to the conformation of globularproteins (a hydrophobic core surrounded by a stabiliz-ing hydrophilic shell) was first described by Khokhlovand Khalatur [1, 2]. The subsequent development ofthis concept revealed a number of intriguing and impor-tant features of mathematical models of these copoly-mers and resulted in the prediction of some effects thatshould be manifested in the behavior of real macromo-lecular compounds with a primary monomer sequencethe same as predicted by calculations for protein-likecopolymers (for a review of these theoretical papers,see [3]).

Along with theoretical studies, successful attemptsat preparative synthesis of some protein-like copoly-mers have been reported [4]. These syntheses wereaccomplished using either chemical modification ofpolymeric precursors [5] or copolymerization [6–9] orcopolycondensation [10] of monomer pairs differing inhydrophilic/hydrophobic characteristics. In particular,by free radical copolymerization of

N-

vinylcaprolac-tam (NVCL) and

N-

vinylimidazole (NVIA) in water ata temperature above the phase separation point of thereaction system (precipitation polymerization), we pre-pared copolymers that had the most pronounced pro-tein-like properties [6, 9, 11] as compared with otherknown samples [5, 7, 8, 10]. It was shown that the targetprotein-like macromolecules were formed only in adefinite rather narrow range of initial NVCL : NVIAratios and that the process yielded a mixture of polymerfractions that differed in water solubility at elevatedtemperatures. They were called thermally precipitating(tp) and thermally nonprecipitating (soluble, ts) frac-

tions. In view of its physicochemical characteristics,the latter was identified as consisting of protein-likemacromolecules [9, 11].

Since these copolymers contained NVIA units,which represent an organic base able to catalyzehydrolysis of ester substrates, as has been shown inrelation to other NVIA-containing polymers [12, 13],we decided to test the resulting protein-like ts fractionof NVCL/NVIA copolymer for this type of catalyticactivity. It was highly important to elucidate the depen-dence of catalytic properties (if detected) on the confor-mational state of copolymer macromolecules in solu-tion, which is determined by the system temperature.Indeed, it was found previously [9, 11] that, below atemperature of about 38

°

C, the macromolecules of thisfraction occur in an aqueous medium in a slightlyordered coil conformation, while, above this tempera-ture, they undergo a transition to a globular protein-likeconformation with a compacted hydrophobic core sur-rounded by a loose polar hydrophilic shell. In addition,it was pertinent to compare the results with the catalyticactivity of the ts fraction of a random (non-protein-like)copolymer resulting from copolymerization of thesame monomer pair in solution at a temperature belowthe phase separation point of the reaction system [9].Note that no studies of this type have been carried outpreviously with protein-like copolymers soluble over abroad temperature range, known works being con-cerned with either NVIA-containing crosslinked gels[12] or with random copolymers aggregating on heat-ing above the lower critical solution temperature [13,14].

The NVCL and NVIA copolymers were preparedby a procedure described in detail previously [9]. Poly-merization was carried out in 10% aqueous dimethylsulfoxide at two temperatures, 25

°

C (homogeneoussolution polymerization) and 65

°

C (heterogeneouspolymerization in which the reaction mixture becameturbid as soon as 1–2 min after addition of the initiator).The overall concentration of the monomers was0.35 mol/l and the NVCL : NVIA molar ratio was

Catalytic Properties of the Protein-Like Copolymerof

N-

Vinylcaprolactam and

N-

Vinylimidazole in the Hydrolysis of an Ester Substrate

V. I. Lozinskii, I. A. Simenel, and

Academician

A. R. Khokhlov

Received April 19, 2006

DOI:

10.1134/S0012500806100028

Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow, 119991 Russia

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CATALYTIC PROPERTIES OF THE PROTEIN-LIKE COPOLYMER 171

85 : 15 because these conditions were shown previ-ously [9] to ensure the highest yields of the ts fractions.The ammonium persulfate–

N

,

N

,

N

',

N

'-tetramethylethyl-enediamine redox pair was used as the initiator. Aftercompletion of the process, the monomeric and oligo-meric components were removed by dialysis through acellulose film against deionized water and the purifiedpolymeric products were freeze-dried. The resultingcopolymer was dissolved again in water and centri-fuged hot at 65

°

C and 8000 rpm for 30 min. The super-natant, containing the ts fraction, was decanted, cooled,and freeze-dried, while the precipitate, representing thetp fraction, was cooled, dissolved in water, and alsofreeze-dried (this fraction was not used in this work totest the copolymer catalytic activity because this poly-mer precipitates above

~34°C

and spectrophotometricmonitoring of hydrolysis of the chromogenic substrateis impossible). To designate the synthesis temperaturewhere the fractions were formed, below, superscriptsare used; ts

65

is the thermally soluble fraction of theNVCL/NVIA copolymer having, as mentioned above,a protein-like primary sequence of monomer units andts

25

is the thermally soluble fraction of theNVCL/NVIA copolymer having a non-protein-likerandom distribution of the NVCL and NVIA unitsalong the chain. Some physicochemical characteristics(published data [9, 11]) of these fractions are summa-rized in the table.

The catalytic activities of the ts

65

and ts

25

fractionswere studied in relation to hydrolysis of

p

-nitrophenylpropionate (NPP) at 25 or 50

°

C in two buffer systems:0.05 M

N-

(2-hydroxyethyl)piperazine-

N

'-(2-ethane-sulfonic acid) (HEPES)–NaOH with pH 7.3 and 8.2.

p

-Nitrophenyl propionate was synthesized inYu.N. Belokon’s laboratory (Nesmeyanov Institute ofOrganoelement Compounds, Russian Academy of Sci-ences) and kindly provided for our study; this is a typi-cal water-soluble substrate used to determine lipaseactivities [15]; i.e., if catalytic action is detected, thesecopolymers could be considered as synthetic models ofsuch hydrolases. Experiments were performed usingsolutions with an initial NPP concentration of0.4

µ

mol/mL. The copolymer concentration was variedfrom 0.033 to 0.130 mg/mL, or from 0.25 to1

µ

mol/mL, relative to the content of imidazole groups.On the one hand, this allowed the experiments to beconducted at NPP/

N-

alkylimidazole (in the polymerchain) molar ratios equal to those used in the previousstudy [13] in order to compare the results, but, on theother hand, the concentrations we used were apprecia-bly (15–60 times) lower.

Figure 1 shows the curves for variation of the absor-bance at 348 nm (absorption of the

p

-nitrophenolateformed) during incubation of the solutions (pH 8.2) at25

°

C (i.e., below the conformation transition tempera-ture of the ts

65

sample) or at 50

°

C (knowingly abovethe conformation transition temperature of this frac-tion). Decomposition of NPP in the presence of the

non-protein-like copolymer (the ts

25

fraction) both at25

°

C (Fig. 1,

2

) and at 50

°

C (Fig. 1,

5

) was found toproceed at approximately the same rate as substratehydrolysis induced merely by the alkaline buffer at thesame temperatures (Fig. 1,

1

and

4

, respectively). Inother words, with relatively low copolymer ts

25

concen-trations (previously [13], this was 2 mg/mL) (see thecaption to Fig. 1), the macromolecules of randomNVCL/NVIA copolymer did not show reliably detect-able esterolytic activity at 25 or 50

°

C, despite thehigher content of NVIA units in the copolymer chaincompared with the ts

65

sample (table), which exhibitedthis activity even at low concentrations (in particular,0.065 mg/mL). Indeed, whereas macromolecules of thets

65

fraction at 25

°

C existing in a disordered coil con-formation catalyzed NPP hydrolysis only slightly, likethe random ts

25

sample (Fig. 1,

3

), after the thermallyinduced conformational transition of ts

65

to the protein-like conformation, substrate cleavage proceededapproximately 2.3 times faster in the presence of thecopolymer (Fig. 1,

6

) than NPP hydrolysis in the alka-line buffer without the copolymer at the same tempera-ture (50

°

C, Fig. 1,

4

). As the temperature was raisedfrom 25 to 50

°

C, the rate of the latter process (in thebuffer) increased ~5.2-fold (cf. curves

1

and

4

, Fig. 1),while, in the presence of ts

65

, the process at 50

°

C was12 times faster (cf. curves

1

and

6

, Fig. 1). This demon-strates the catalytic effect caused by specific folding ofthe copolymer chains where hydrophilic groups of

N-

alkylimidazole were accumulated in the peripheralregions of protein-like polymeric particles (dependingon the concentration, these may be either separate mac-romolecules or soluble micelles consisting of macro-molecular associates [11]). Therefore, as regards theeffect on NPP hydrolysis at 50

°

C, the ts

65

fraction of theNVCL/NVIA copolymer may be regarded as “enzyme-like,” meaning that its macromolecules exhibit clear-cut catalytic activity only in a definite conformation.

Figure 2 shows kinetic curves for NPP hydrolysiscatalyzed by different concentrations of ts

65

at pH 8.2and 50

°

C. An increase in the content of the enzyme-like

Characteristics of the NVCL/NVIA copolymers used in thestudy (published data [9, 11])

Copolymerfraction

Molecularmass*

Molar ratio of monomer

units**

Thermodynamic param-eters of the conformation

transition***

transition tem-perature (maxi-

mum),

°

C

transitionenthalpy,

J/g

ts

25

120000 61.5 : 38.5 – –

ts

65

40000 73.0 : 27.0 38 6.5

* Determined by gel permeation chromatography. ** Found by NMR spectroscopy.*** Measured by high-sensitivity differential scanning calorimetry.

172

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LOZINSKII et al.

polymeric catalyst from 0.033 (Fig. 2,

2

) to 0.065(Fig. 2,

3

) and then to 0.130 (Fig. 2,

4

) mg/mL (or from0.25 to 0.5 and then to 1

µ

mol/mL relative to

N-

alkyl-imidazole units) led to an increase in the reaction rateby factors of 1.5, 2.3, and 3.4, respectively, compared tothat of substrate hydrolysis by the buffer alone. Thus,this concentration dependence also indicates that this isan enzyme-like copolymer. Recall that, under similarconditions, random NVCL/NVIA copolymer, unable toacquire a protein-like conformation (i.e., the

ts

25

frac-tion), had barely any catalytic effect (Fig. 1,

5

).

Since NPP was hydrolyzed at a rather high rate at50

°

C and pH 8.2 (Fig. 1,

4

), it was difficult to elucidatethe effects of low concentrations of polymeric cata-lysts; hence, we studied hydrolysis also in less alkalineHEPES/NaOH buffer at pH 7.3. The results of experi-ments are shown in Fig. 3. In this case, a substantial(4.1-fold) increase in the reaction rate was observed inthe presence of the enzyme-like ts

65

polymer even at25

°

C (Fig. 3,

2

) as compared with NPP hydrolysis bythe buffer alone (Fig. 3,

1

), while the non-protein-likets

25

fraction of random NVCL/NVIA copolymer barelyaffected substrate hydrolysis (this is not shown in Fig. 3to avoid superposition of almost identical curves). Thisresult suggests that ordered regions may exist in thestructures of macromolecules of the protein-like ts

65

fraction also below the conformational transition tem-perature. This is consistent with the conformationalmemory effect predicted theoretically for this type ofcopolymers [1–3, 6], which is specified in this case bythe conditions of synthesis. In addition, the absolute

value of acceleration of NPP hydrolysis by the enzyme-type ts

65

copolymer was markedly greater at pH 7.3than at pH 8.2. Whereas NPP hydrolysis in the bufferwith pH 7.3 increased 5.4-fold (at pH 8.2, 5.2-fold)upon a temperature rise from 25 to 50

°

C (cf. curves

1

and

3

, Fig. 3), in the presence of the ts

65

copolymer, itincreased 21.6-fold (cf. curves

1

and

4

, Fig. 3), and atpH 8.2 a 12-fold accelerating effect was observed (seeabove, the discussion of Fig. 1), although the kinetic

12

3

4

5

6

0 600400200

Time, s

0.2

0.4

0.6

Absorbance at 348 nm

200 400 600

0.2

0.4

0.6

Time, s

Absorbance at 348 nm

1

2

34

0

1

3

5

Absorbance at 348 nm

200 400 600

0.2

0.4

0.6

Time, s

0

2

4

Fig. 1.

Dynamics of

p

-nitrophenol liberation on NPPhydrolysis in a 0.05 M HEPES/NaOH buffer (pH 8.2) at(

1

) 25 and (4) 50°C and in the presence of NVCL/NVIAcopolymers (0.5 µmol/mL relative to the content of N-alkyl-imidazole groups) ts25 at (2) 25 and (5) 50°C and ts65 at(3) 25 and (6) 50°C. Measurements were carried out on aSpekol 221 (Carl Zeiss, Jena, Germany) spectrophotometer;the temperature was maintained by a ED 5 ultrathermostat(Julabo, Germany).

Fig. 2. NPP hydrolysis in a 0.05 M HEPES/NaOH buffer(pH 8.2) at (1) 50°C and in the presence of different concen-trations (relative to the content of N-alkylimidazole groups)of the ts65 fraction of the NVCL/NVIA copolymer: (2) 0.25,(3) 0.5, and (4) 1.0 µmol/mL.

Fig. 3. NPP hydrolysis in a 0.05 M HEPES/NaOH buffer(pH 7.3) at (1) 25 and (3) 50°C and in the presence of thets65 fraction of the NVCL/NVIA copolymer (0.5 µmol/mLrelative to the content of N-alkylimidazole groups) at (2) 25and (4) 50°C. (5) Dynamics of NPP hydrolysis at pH 8.2and 50°C.

DOKLADY CHEMISTRY Vol. 410 Part 2 2006

CATALYTIC PROPERTIES OF THE PROTEIN-LIKE COPOLYMER 173

curve for NPP hydrolysis in the more alkaline buffer(Fig. 3, 5) is located somewhat higher than that for pH7.3 (Fig. 3, 4), which is quite understandable in view ofmore intensive substrate saponification at pH 8.2. Notealso that the esterolytic activity of the thermally solublefraction of the NVCL/NVIA copolymer whose macro-molecules have a protein-like conformation under theseconditions is observed at much lower concentrations ofthe polymeric catalyst than for alternating NVIA-con-taining copolymers known from the literature [13, 14],for which the activity jump upon thermoprecipitationwas associated with the formation of hydrophobic–hydrophilic interfaces between the polymeric and liq-uid phases, which was favorable for specific adsorptionof amphiphilic substrate molecules. For the enzyme-like NVCL/NVIA copolymer studied here (the ts65fraction), acceleration of the hydrolysis of an ester-typesubstrate is due most likely not only to the formation ofhydrophobic–hydrophilic interfaces (apparently, at thelevel of globules of separate macromolecules) but alsoto local accumulation of catalytically active N-alkylim-idazole units in the peripheral regions of the protein-like polymer particles.

ACKNOWLEDGMENTSThis work was supported by the fundamental

research program of the Division of Chemistry andMaterials Science of the RAS “Design and Study ofNew-Generation Macromolecules and Macromolecu-lar Structures” for 2006–2008 and the Federal Agencyfor Science and Innovation of the Russian Federationwithin the framework of the program “Studies andDesign along Priority Lines of the Development ofScience and Engineering” for 2002–2006 (projectno. 2005–ZhS–12.4/002).

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