Chemistry and Physics of Lipids, 62 (1992) 45-54 Elsevier Scientific Publishers Ireland Ltd.

45

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy M i s a k o W a k i t a a, Y o s h i h i r o K u r o d a a, Y a s u h i r o F u j i w a r a b a n d T e r u m i c h i N a k a g a w a a aFaculty of Pharmaceutical Sciences, Kyoto University and bKyoto Pharmaceutical University, Kyoto (Japan) (Received January 2nd, 1992; revision received March 16th, 1992; accepted March 31st, 1992)

Conformations of dibucaine and tetracaine in small unilameilar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in IH nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.

Key words: local anesthetics; lipid bilayer; nuclear Overhauser effect; lH-NMR; molecular dynamics

Introduction

Local anesthetics are known to cause their anesthetic action by affecting the function of the sodium channel in nerve axonal membranes [1]. However, whether this action is a result of a specific anesthetic-protein interaction [2,3] or a non-specific perturbation to the lipid bilayer structure [4,51 is still an unsolved problem [1]. Interactions between anesthetics and enzymes [6], membrane-bound acetylcholine receptor [7], or membrane skeleton proteins [8] which may associate with the function of the sodium channel may also be relevant to anesthesia. Even if this mechanism is responsible for local anesthesia, interactions with membrane lipids appear to be a most important first step for the anesthesia; this is because it is generally accepted that the anesthetics exert their effects when present on the cytoplasmic Correspondence to: Y. Kuroda, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan.

side (i.e., inner-side) of excitable membranes, requiring outer- to inner-side transbilayer movement of the drug [9]. Thus, much attention has been devoted to clarify the interactions between anesthetics and lipids from various points of view [10-12]. However, no attention has been paid on the conformation of a local anesthetic in membranes. Recently, Greenberg and Tsong have isolated a protein which has high affinity for local anesthetics, by employing quinacrine as a fluorescent local anesthetic probe. They showed that the protein provides a local anesthetic receptor site in axonal membranes [13,14]. If this is true, the molecular structure of a local anesthetic in membranes, not in a buffer solution, is expected to play an important role in causing anesthesia, as has been suggested by Sargent and Schwyzer for the cases of membrane-catalyzed peptide-receptor interactions [15]. The present work has been undertaken to inquire into the conformations of dibucaine and tetracaine in lipids. To investigate the conformations of the drugs in a lipid matrix

0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

46 would also lead to better understanding of the perturbation to the lipid bilayer structure by the anesthetic molecules as well as to the clear-cut concept of the anesthetic-receptor protein interactions. Materials and Methods

Materials Egg yolk L-~-phosphatidylcholine (egg PC), dibucaine hydrochloride and tetracaine hydrochloride were purchased from Sigma and used without further purification. Preparation of sample solution Preparation of single bilayer vesicles (40 mM) were made as described previously [16] except that 0.1 M phosphate buffer in 2H20 (pH 6.7, meter reading) was employed. The ionic strength of the solution was found from calculation to be 0.6. A weighed amount of a drug dissolved in the buffer was added to the solution of the pre-formed vesicles; concentrations of the drugs were 40 mM. Measurements The IH-NMR experiments were carried out on a Bruker AM-600 (600 MHz) spectrometer or on a Shimadzu/GE Omega-500 (500 MHz) spectrometer. Phase-sensitive NOE and chemical exchange correlated spectra (NOESY) were measured by using a standard 90° three-pulse sequence and with a TPPI phase-sensitive mode [17]. Rotating-frame NOE experiments (ROESY) were performed as described by Bax et al. [18]. The strength of the spin-lock field was 2630 Hz 0r/2 = 95 ~s) and the rf carrier was positioned at 4.8 ppm. The chemical shifts were referenced to the residual H2HO protons (4.8 ppm). Ambient probe temperature was 27°C. Molecular dynamics calculations All of the modeling studies were conducted with Molecular Simulations' NMRgraf software [19] running on a Silicon Graphics Iris 4D/25 computer. A starting structure of dibucaine was construtted by using an organic molecule builder equipped with the software; we considered both trans- and cis-forms of the amide of dibucaine. A

restrained molecular dynamics method was used to calculate the energetically minimum structures of dibucaine. It incorporates NOE distance constraints into the mechanical force fields of the dibucaine molecule which include bond stretch, angle bend, torsion, electrostatic, van der Waals and hydrogen bond energies [20]. All of the NOE distance constraints were assumed to be in the range 2.0-4.0 A. Results

Two-dimensional R O E S Y and N O E S Y spectra o[ dibucaine Figure 1 shows ROESY (A) and NOESY (B) spectra of dibucaine hydrochloride (40 mM)-egg PC (40 mM) solution; cross-peaks shown are between aromatic protons and the ones which resonate higher field than the residual H2HO protons at 4.8 ppm. We assigned the proton resonances based on our previous work [16]. In the ROESY spectra all the cross-peaks were outof-phase to the diagonal peaks, while in the NOESY spectra all the cross-peaks were in-phase to the diagonal peaks. The former observation means that the ROESY cross-peaks are due to direct dipole-dipole interactions, while the latter indicate that the mobility of dibucaine is highly reduced on account of interaction with the lipids. Thus, the cross-peaks in the NOESY spectra originate not only from direct dipole-dipole interaction but also from spin diffusion [21]. However, we found that this spin diffusion remarkably facilitated assignment procedures for resonances due to the dibucaine molecule. For example, starting from the resonance due to ~b3, we could easily follow resonances due to the butoxy group of dibucaine. Also, starting from the same resonance (~3), assignments for resonances arising from the polar side-chains could easily be made. Observed NOE cross-peaks of dibucaine in liposomes are summarized in Table I. Conformations of dibucaine in a lipid matrix We considered four kinds of conformation, A-D, of dibucaine which satisfy the observed NOE distance constraints in Table I. Among these, Fig. 2 shows schematic drawings of the con-

47

¢5 (1'8

DIBUCAINE ,~HCH2CH2N(CH2CH3)2.HCI

7~OCH2CH2CH2CH

cH3-/

o

A

i

NH*(C-CH3)2 CH2CH3~ < OCH2CH~~

•I .0

I.0

"2.0

-2.0

-3.0

3.0

!

i

|

NH'(cH2-C)2- ~ CONHCH2 f

CH2NI-~ OCH2

3

*0

"40

-

&O

'0

p: . - - - - - ' '

'

o...

'

a'O

.

.

.

.

.

7~0

'

'

'

OPM

......

810. . . . . .

70

PPM

Fig. 1. 600 MHz ROESY (A) and NOESY (B) spectra of dibucaine-egg PC vesicles solution, Spin-lock and mixing times were 25 and 50 ms, respectively. The inset shows the structure of dibucaine hydrochioride and the numbering scheme of the aromatic ring.

formations, A, B and C, representing a NOE connectivity by a dashed line. The conformation D, not shown here, directs both the diethylamino group and the butoxy group towards the ¢3 proTABLE I Observed NOE connectivities of dibucaine and tetracaine in egg PC vesicles associated with orientations of the side-chains relative to the aromatic rings. Ring proton

Side-chain/ring proton

Dibucaine

03, ¢5, ~s 03, ~5 03, ¢s

05

NH+(C-CH3)2 NH+(CH2-C)2 CONHCH2* CH3 OCH2CH2* ¢s

Tetracaine

02,6

NH+(CH3)2

03,5

CH2NH CH3 NH+(CH3)2

*Resonances of starred protons are discussed.

ton of the quinoline ring. The dimerized form of this conformation, however, seems to be the most unstable one in a lipid matrix, as will be described later. Evidently any one of the conformations cannot satisfy all the observed NOE distance constraints (Table I) at once. Moreover, obviously any type of conformation considered here cannot satisfy the NOE distance constraints between ¢8 and ~he methyl and methylene protons of the diethylamino group and also between es and ¢5 (Table I); these NOEs appear to be due to intermolecular dipole-dipole interactions in origin. Thus, in order for these NOE distance constraints to be satisfied, we had to consider association of dibucaine molecules. As the simplest mode of association, it is conceivable that the two dibucaine molecules associate with each other facing the two quinoline tings as shown schematically in Fig. 3A-C. This is a so-called stacking interaction between the aromatic rings. Figure 3A,B and C represent, respectively, association of dibucaine molecules having conformations A, B and C in Fig. 2; we denote these dimers as A-A, B-B and CC. In Fig. 3, the polar side-chain which includes

48

(a)

~

"

(b)

2

H2

CHs C H 2

(c) ~2'~x,,0

H C

. H2 ~'C

CHs CH2

u b. ,,u

CH, H

H s

eT " ~ - 0

s

' sH,....H~C:/ 7

3

H~C H~C

"CH,

H 3g "H~c

..6

A C Fig. 2. Possible structures of dibucaine in egg PC vesiclessolution: (a), conformation A; (b), conformation B; (c), conformation C. NOE distance constraints are shown by dotted lines.

the diethylamino group is drawn as a line which possesses a positive sign at its terminal position and the butoxy group as a line which includes OMe; the solid line denotes anterior and the dotted line posterior molecules. These dimers can satisfy the above-mentioned three N O E distance constraints. In A-A, the cationic diethylamino group and polar oxygen and nitrogen atoms direct upwards and the hydrophobic aromatic ring and aliphatic butyl group downwards. Accordingly, this type of association appears to be most stable in a lipid matrix. The B-B type o f association appears to be less stable than A-A, because the hydrophobic aromatic ring is directed towards the

Ca) +

f

(c)

(b) $

lipid-water interface. O n the basis of the same reasoning, the C-C type association may also bc unstable in lipids.W e could further consider A-B, A-C and B-C types of association. However, these are unlikely, because these aggregates direct the cationic diethylamino group of the two dibucainc molecules opposite to each other, making no clearcut distinctionbetween the polar and hydrophobic moieties of dibucaine. A n example for the A-B type of association is shown in Fig. 3D. Based on a similar reasoning, a D - D type of association, which directs both the polar and hydrophobic side-chains to the same direction, may be the most unlikely case in a lipid matrix.

(d)

Jr

0.~---/-. 0 A-A

B-B

C-C

A-B

Fig. 3. Schematic drawings for the association of dibucaine in egg PC vesicles. Stacking interactions between the aromatic rings are considered here; the solid line denotes anterior and the dotted line posterior molecules. The dimars shown in (a), (b) and (c) are constructed from the two moleculeshaving conformations A, B and C, respectively,shown in Fig. 2; the dimer shown in (d) is constructed from the moleculeshaving conformations A and B.

49

Two-dimensional ROESY and NOESY spectra of tetracaine Figure 4 shows ROESY (A-C) and NOESY (D-F) spectra of tetracaine hydrochloride (40 mM)-egg PC (40 mM) solution observed at spinlock (ROESY) and mixing (NOESY) times of 50, 100 and 200 ms; contours shown are between aromatic protons of tetracaine and those of the same molecule which resonated higher field than 4

ppm. In the NOESY spectra, phases of all the cross-peaks were in-phase to the diagonal-peaks and the observed NOEs were overwhelmed by spin diffusion phenomena. Here again, these spin diffusions facilitated the assignments of tetracaine resonances which are buried within very broad resonances of the lipids. For example, we could trace the butylamino protons starting from the aromatic $3,5 protons of tetracaine. In the

TETRACAINE CH3CH2CH2CH2NH4(~,COOCH2CH2

¢2.6

3

~s

N (CH3) 2 ' HCI

2

2 _ _

CH3

---

[]

i.

. . . . .

:':_:_,:~

~_-. - - - , . . . . . .

• ' ,

NH'(

.

.

.

.

.

" "

,

" :

,

. .

.

,!:. 8.5

7.5

6.5

PPM

CH3 0 .......... -,..... CH3CH~ . . . . . -~;~_ : - : - - - ' _ - - - ~ ' _ ' ~ - - _ "

, C1"6CH2NH

:___

8.5

•

7.5 PPNI

6.5

E " " - 1.0

--*

. . . .

B.5

I ~

.......

7.5 PPM

6.5

I-F 1.0

--

-¢

........

II

_°.

:I

LO-

.

.

.

.

.

.

.

.

.

.

.

.

.

......

- 3.0-

........

C H2NH,, ,,-~: -_f- . . . . . . . . . . . . ~ . . . . . CH2NI"I* "~ .... ~:.... '---, . . . . . : - - : - ,,.,-d... :.. ' ..'. , ...... ~..., s"i . . . . . . .

"7'.s " " ' " ' 6 '~. . . . P~d

s. . . . . . . . .

'¢,5' . . . . . . . PRM

6~i

.g'"""

?!s. . . . . l:'l~

6'.g ' ' '

Fig. 4. 500 MHz ROESY (A-C) and NOESY (D-F) spectra of tetracaine-egg PC vesicles solution. The inset shows the structure of tetracaine hydrochioride and the numbering scheme of the aromatic ring. Spin-lock times were: (A), 50 ms; (B), 100 ms; (C), 200 ms. Mixing times were: (A), 50 ms; (B), 100 ms; (C), 200 ms.

50

ROESY spectra, phases of all the observed crosspeaks were out-of-phase to those of the diagonal peaks, except that the phase of the cross-peak between 03,5 and NH+(CH3)2 changed from out-ofphase at the spin-lock time of 50 ms to in-phase at the spin-lock times longer than 100 ms. This alteration in the phase of the cross-peak can be ascribed to the increased contribution of spin diffusion [22]. Observed NOE cross-peaks in the ROESY spectra are summarized in Table I. Conformations of tetracaine in a lipid matrix The NOE distance constraints in Table I propose two kinds of conformation, A and B, for tetracaine as shown in Fig. 5. These conformations satisfy the NOEs between 02,6 and NH+(CH3)2 and between ~3,5 and CH 3 of the butylamino group. However, the remaining two NOEs cannot be satisfied on account of the restraints required from the molecular structure of tetracaine. Again, as in the case of dibucaine, there remained two NOEs, that is, between 02,6 and CH2NH and between 03,5 and NH+(CH3)2. These can be considered to be due to intermolecular dipole-dipole interactions. In order for these NOEs to be satisfied, we considered the following types of stacking interactions between the aromatic rings of tetracaine, that is, A-A and B-B (Fig. 6). Here, a line with a positive sign means a cationic dimethylamino side-chain and a line with -N-Me means a butylamino group; a dotted line denotes the side-chain originated from a posterior molecule. Although both A-A and B-B can satisfy the above-mentioned intermolecular NOEs, the A(a)

H2

(b)

c-c-..H+, H, , 7 OH, 0,,~ 0 H3C/ C H".''H" "~i"~5"H / ..N-.. FhC .~/,C,2 H

hj

A

H2

0:~, 0

H3C /

H~H-"H3C N,., \ H" C~. /CH, H2 ~2

B

Fig. 5. Possible structures of tetracaine in egg PC vesicles solution: (a), conformation A; (b), conformation B.

(a)

(b)

+

+

A-A

(c)

+

+

B-B

+

+

A-B

Fig. 6. Schematic drawings for the association of tetracaine in egg PC vesicles. Stacking interactions between the aromatic rings arc considered here; the solid line denotes this side and the dotted line backward molecules. The dimers shown in (a) and (b) are constructed from the two molecules having conformations A and B, respectively, shown in Fig. 5; the dimer shown in (c) is constructed from the molecules having the conformations A and B.

A type of association appears favorable in lipids, because this sort of association can separate the hydrophilic and hydrophobic moieties of the aggregate distinctly. Using the same reasoning, the A-B type of association shown in Fig. 6C also appears less stable than A-A. Molecular dynamics calculations for dibucaine and tetracaine and their dimers In order to obtain energetically minimum conformations which satisfy NOE distance constraints for the models schematically considered above, we performed molecular dynamics calculations. More importantly, the calculations were made to examine to what extent the association of dibucaine could reduce the strain energy of the corresponding monomer molecule. Firstly, we performed calculations for a monomer by incorporating only the intramolecular NOEs into the energy minimization cycles. Secondly, for the corresponding dimer, calculations included incorporation of both intra- and intermolecular NOEs and refinement of all the coordinates of the dimer. The calculated energies of both the trans and cis forms of the amide linkage of dibucaine are summarized in Table II. The computer graphics pictured for energetically minimized dibucaine monomers and dimers are shown in Figs. 7-9, respectively, for the cases of the trans-amide in conformations A, B and C. In the case of tetracaine, we calculated only

51 T A B L E II Calculated mechanical strain energies of trans- and c/s-amides of dibucaine and their dimers. Type of conformer A B C D

C/s-amide a

Trans-anfide a

Monomer

Dimer

AE b

Monomer

Dimer

AE b

104.3 104.9 95.3 110.3

178.6 181.4 172.6 201.9

-30.0 -28.4 -18.0 -18.7

112.7 103.5 97.6 91.4

175.3 219.4 240.6 170.0

-50.1 +12.4 +45.4 -12.8

aEnergy (kcal/mol). bAE = the stabilization energy, which is calculated by subtracting twice the energy value of a m o n o m e r from the energy value of a dimer.

for conformation A (Fig. 5), because no well defined distinction could be found between conformations A and B. The calculated energies of monomer, dimer and AE were 63.2, 113.9 and -12.5 Kcal/mol, respectively; AE is the difference between the energy of a dimer and twice the energy of the monomer and means stabilization energy as a result of association. The computer graphics pictured for the monomer and dimer are shown in Fig. 10. Inspection of Table II indicates that the association of dibucaine really reduced the strain energy except for the cases of the cis form of conformations B and C; this is reflected in the stabilization energy AE. As for tetracaine, again, association can be stabilized by the stacking interactions between two aromatic rings. Discussion

It is known that local anesthetics such as the amines dibucaine and tetracaine form micelles in solution, since they are amphiphilic molecules; their cmc's are in the range of 30-60 mM [23-25]. These critical micelle concentration (cmc) values decrease with increasing ionic strength of the solutions; for example, the cmc of dibucaine at the ionic strength of 0.6 can be roughly estimated to be 15 mM [23]. In the present experimental conditions, if we assume percentages of the bound drugs to be 40% for both dibucaine and tetracaine [16], the concentrations of the drugs in the buffer solution (24 mM) can exceed the cmc; thus, it is con-

ceivable that both dibucaine and tetracaine exist as micelles. In contrast, the present NOE data show that these anesthetics reside in lipids as a dimer. Although rather concentrated sample solutions (40 mM) may be partly responsible for this association, to form a dimer in a lipid matrix appears to be attributable to their chemical properties. According to the present molecular dynamics calculations, the stabilization energy that may lead to the dimerization is contributed to not only from the van der Waals forces between two aromatic rings but also from the electrostatic and in some cases from the hydrogen bond forces. The formation of a hydrogen bond was noted in the cases of conformations B and C of dibucaine having a trans-amide linkage and also in tetracaine. However, it should be remembered that the present molecular dynamics calculations are for molecules in a vacuum. Thus, in order to discuss more rigorously the stabilization energy necessary to form a dimer in a lipid matrix, we should include the interactions with the lipid and also with the water molecules which may surround the anesthetic molecules. These calculations are currently being designed. According to Hille, local anesthetics bind strongly to resting and inactivated forms of the Na channel [26]. On the other hand, Greenberg and Tsong reported that they purified an integral protein with a molecular mass of 16 kDa which provides a receptor site for a local anesthetic in mammalian axonal membranes [13,14]; this pro-

52

Fig. 8. As in Fig. 7, but with conformation B.

Fig. 7. Computer graphics view of the dibucaine having conformation A and the trans form of the amide linkage; the upper picture shows a monomer and the bottom picture its associated state.

tein differs from that of the Na channel, Although, we cannot judge these conflicting views at this momeat, the dimerized form as revealed from the present NOE data can be an active (effective) three-dimensional structure of the amino local anesthetics which can block action potentials in

excitable membranes. Sargent and Schwyzer have considered that the conformation and orientation of a ligand in a lipid bilayer is an important threedimensional structure just before it binds with the receptor [15]. Interestingly, all of the clinically useful local anesthetics possess an aromatic ring; this is especially true for the amino local anesthetics. Consequently, all these anesthetics can perform stacking interactions by themselves which are reinforced by intermolecular electrostatic and hydrogen bond forces at their amine

53

®

®

Fig. 9. As in Fig. 7, but with conformation C. Fig. 10. Computer graphics view of the tetracaine having conformation A; the upper picture shows a monomer and the bottom picture its associated state.

and intermediate ester or amide chain portions. It seems that such a dimerized anesthetic as shown in Figs. 7 - 1 0 is anesthetizing the N a channel, locating itself at an amphiphilic part o f the N a channel or at the receptor protein.

Acknowledgements We thank Dr. Matashige O y a b u o f Shimadzu Co. for the measurements o f 500 M H z N M R spectra. We also thank Dr. J.W. Hare o f Simulation

54

Technology Inc. for dynamics simulations.

performing

molecular

References l

G.R. Strichartz and J.M. Ritchie (1987) in: G.R. Strichartz (Ed.), Local Anesthetics, Spdnger-Verlag, New York, pp. 21-52. 2 C.D. Richards, C.A. Keightley, T.R. Hesketh and J.C. Metcalfe (1980) Prog. Anesthesiol. 2, 337-351. 3 B. Hille (1980) Prog. Anesthesiol. 2, 1-6. 4 A.G. Lee (1976) Nature 262, 545-548. 5 J.R. Trudell (1977) Anesthesiology 46, 5-10. 6 G.C. Kresheck, A.B. Adade and G. Vanderkooi (1985) Biochemistry 24, 1715-1719. 7 S.G. Blanchard, J. Elliott and M.A. Raftery (t979) Biochemistry 18, 5880-5885. 8 Y. Srinivasan, L. Elmer, J. Davis, V. Bennett and K. Angelides (1988) Nature 333, 177-180. 9 T. Narahashi, D. Frazier and M. Yamada (1970) J. Pharmacol. Exp. Ther. 171, 32-44. 10 M. Auger, I.C.P. Smith and H.C. Jarrell (1989) Biochim. Biophys. Acta 981,351-357. 11 C. Gutierrez-Merino, A. Molina, B. Escudero, A. Diez and J. Laynez (1989) Biochemistry 28, 3398-3406. 12 A. Seelig, P.R. Allegrini and J. Seelig (1988) Biochim. Biophys. Acta 939, 267-276.

13 14 15 16 17 18 19 20 21 22 23 24 25 26

M. Greenberg and T.Y. Tsong (1982) J. Biol. Chem. 257 8964-8971. M. Greenberg and T.Y. Tsong (1984) J. Biol. Chem. 259 13241-13245. D.F. Sargent and R. Schwyzer (1986) Proc. Natl. Acad Sci. U.S.A. 83, 5774-5778. Y. Kuroda and Y. Fujiwara (1987) Biochim. Biophys. Ac. ta 903, 395-410. G. Bodenhausen, H. Kogler and R.R. Ernst (1984) J Magn. Reson. 58, 370-388. A. Bax and D.G. Davis (1985) J. Magn. Reson. 63 207-213. Molecular Simulations, Inc., 796 North Pastoria Avenue Sunnyvale, CA 94086, USA. S.L. Mayo, B.D. Olafson and W.A. Goddard III (1990) J Phys. Chem. 94, 8897-8909. Y. Kuroda and K. Kitamura (1984) J. Am. Chem. Soc 106, 1-6. A. Bax, V. Sklenar and M.F. Summers (1986) J. Magn Reson. 70, 327-331. D. Attwood and P. Fletcher (1986) J. Pharm. Pharmacol 38, 494-498. E.M. Johnson and D.B. Ludlum (1969) Biochem. Phar macol. 18, 2675-2677. M.S. Fernandez (1981) Biochim. Biophys. Acta 646 27-30. B. Hille (1984) Ionic Channels of Excitable Membranes Sinauer Associates Inc, Sunderland, Massachusetts.